Isolation and Characterization of Human Tissue Resident Memory T cells

Abstract

The majority of immune cells in the human body exist within the tissues rather than in the circulation. Nevertheless, in humans the majority of our knowledge of the human immune system is biased towards the characterization and understanding of circulating immune cell populations because the latter are readily sampled, whereas cells in tissues are difficult to obtain and/or are limited to single sites of disease. Tissue-resident cells differ from circulating due to tissue specific niche adaptations that influence their phenotype and function. For instance, T cells in tissues, resident memory (T_RM cells), exhibit tissue specific properties that allow optimal protection from infection due to an acquired ability to coordinate rapid and efficacious pathogen clearance. Thus, to fully understand T cell responses in various pathological conditions one must focus on the properties of T_RM cells and how they have been shaped by their environment. Moreover, one must sample and analyze T cells from multiple tissues, optimally from the same individual, to determine how infectious, cancer, or autoimmune challenge is affecting homeostatic function. Our longstanding collaboration with the organ procurement organization, LiveOnNY, provides unique access to multiple lymphoid, mucosal, and peripheral tissues from organ donors where consent for research use has been obtained. These samples have enabled characterization of human tissue resident memory T cells and other immune cell types across a variety of tissues. Concomitant with this endeavor, we developed and refined a series of methodologies critical for extracting immune cells from tissue for the purpose of phenotypic and mechanistic interrogation. Here, we describe our optimized protocols for processing select human tissues and the requisite coordination and considerations for their maximal yield and tissue quality.

Basic Protocol 1: Isolation of immune cells from blood rich sites (Spleen [SPL], Blood [BLD], Bone Marrow [BOM])

Basic Protocol 2: Isolation of immune cells from lymph nodes, tonsils, and thymus (Iliac Lymph Nodes [ILN], Lung lymph nodes [LLN], Mesenteric Lymph Nodes [MLN], Colonic Lymph Nodes [CLN], Hepatic Lymph Nodes [HLN], Tonsils [TON], Thymus [THY])

Basic Protocol 3: Isolation of immune cells from the lungs (Lung [LNG], Bronchioalveolar lavage [BAL])

Basic Protocol 4: Isolation of immune cells from the intestines (Jejunum Epithelial Layer [JEL], Jejunum Lamina Propria [JLP], Colon Epithelial Layer [CEL], Colon Lamina Propria [CLP])

Basic Protocol 5: Isolation of immune cells from the liver (Liver [LVR])

Basic Protocol 6: Immune cell staining for flow cytometry

Introduction

T cells are pivotal components of the adaptive immune system in that they provide long-term protection from pathogens and cancer. Whereas B cells release their hallmark receptors to neutralize targets, T cells directly engage with targets through their T cell receptors or in response to the proximal cytokine milieu. Consequently, to provide robust cellular immunity, T cells must be present in tissues throughout the body and to have characteristics enabling responses suited to particular environments. Along these lines, both CD4 and CD8 T cell subsets have been described that tend to home to and populate certain tissue and circulatory sites: central memory T cells (T_CM) home to and predominate in lymphoid tissue, effector memory T cells (T_EM) home to and predominate in non-lymphoid tissues, and effector memory T cells re-expressing CD45RA (T_EMRA) predominate in the circulation (Gattinoni et al., 2011; Kumar, Connors, & Farber, 2018; Sallusto, Lenig, Förster, Lipp, & Lanzavecchia, 1999). In addition, we and others have described a non-recirculating subset of T_EM cells which are retained within tissues as tissue resident memory T cells (T_RM) (Masopust et al., 2010; Teijaro et al., 2011). These T_RM provide robust local immune responses within tissue by utilizing various mechanisms such as: direct effector function, the recruitment of additional immune cells, and enhancement of effector cell priming in tissue-associated lymph nodes (Paik & Farber, 2020; Schenkel & Masopust, 2014; Szabo, Miron, & Farber, 2019). T_RM cells adapted to a specific tissue of residence can be quite distinct from T_RM cells adapted to other tissues (Connors et al., 2023; Poon et al., 2023).

The study of human T_RM has remained a significant challenge due in no small part to the availability of human tissue for research and the variety of tissues within an individual study. As such, most human T cell analyses rely on collection of cells from the blood, which does not always recapitulate the response of cells localized within tissue (Kumar et al., 2017). Over 14 years ago, we established a long-standing collaboration and research protocol with LiveOnNY, an organ procurement organization (OPO) for the New York metropolitan area and other OPOs that allows us to obtain multiple tissues and organs from brain dead (deceased) organ donors (Sathaliyawala et al., 2013; Thome et al., 2014; Turner et al., 2014). This has been enabled by next-of-kin consent for the use of tissue in research (Carpenter et al., 2018; Farber, 2021). We have made use of this resource to profile the human immune system across space (blood and different anatomic sites, Fig 1) and over age, with a particular focus on characterizing T cells (circulating and tissue-resident) across the body.

Figure 1:

Example diagram of donor tissue sites.

Tissue Abbreviations: BLD - Blood, BOM - Bone Marrow, SPL - Spleen, ILN - Iliac Lymph Nodes (LNs), CLN - Colonic LNs, LLN - Lung LNs, HLN - Hepatic LNs, MLN - Mesenteric LNs, BAL - Bronchioalveolar Lavage, LNG - Lung, JEL - Jejunum Epithelial Layer (EL), JLP - Jejunum Lamina Propria (LP), aCEL - ascending Colonic EL, aCLP - ascending Colonic LP, LVR - Liver. Color Coding: Red - Protocol 1, Yellow - Protocol 2, Blue - Protocol 3, Green - Protocol 4, Orange - Protocol 5. Image generated using NIH Bioart Source (https://bioart.niaid.nih.gov/)

The acquisition of multiple tissues including lymphoid organs (bone marrow, thymus, spleen, tonsils, and different lymph nodes), mucosal/barrier sites (lungs, intestines), among other sites (e.g., liver) allows for precise definition of the impact tissue environment has on immune cells within an individual and between different individuals (Poon et al., 2023; Szabo et al., 2019). To perform these analyses, we have developed several protocols (Steven, Szabo, & Lam, 2023; Wells, Dogra, et al., 2023; Wells & Szabo, 2023; Wells, Szabo, & Lam, 2023; Wells, Szabo, Lam, & Poon, 2023; Wells, Szabo, & Ural, 2023) to extract and ensure viability of all major immune cell subsets (including T cells, B cells, NK cells, ILCs, Macrophages, Monocytes, Dendritic cells, and Granulocytes) from various tissues. These protocols and the strategic planning necessary to effectively execute them are outlined herein. Furthermore, we provide a basic flow cytometry staining protocol and an example of a spectral cytometry panel for assessing human tissue T cell subsets, differentiation, and functional states.

Cautions: Use of organ donor samples

The use of organ donor tissue is not considered ‘Human Subjects’ work because the samples are obtained from deceased, not living individuals. Our exclusion criteria for the organ donor protocol excludes individuals with active infection including SARS-CoV-2, Hepatitis, or HIV, as well as cancer, and chemotherapy. These criteria are to ensure that we are profiling the human immune system in an overall healthy state. The tissues are obtained at the time of acquisition of organs for life-saving transplantation, enabling functional and high dimensional profiling of live cell populations within these samples.

The tissues are brought back to the laboratory within hours of acquisition and processed by laboratory personnel using the protocols outlined below. We use universal precautions for the handling of all human samples, including biosafety level 2, use of biosafety cabinets, and personnel handling these samples have standard personal protective equipment (PPE) including laboratory coats, eye protection, gloves, and sleeve protectors. All waste is disposed of in biohazard waste containers and pipettes are disposed in sharps containers. Further, all tissue processing and handling occurs within biosafety cabinets.

Strategic Planning

The major challenge in processing multiple human samples is inherent in the large sample size and volume of such samples—much larger than those of mouse tissues. We have optimized human tissue processing techniques and have developed site-specific protocols for obtaining single cell suspensions from such tissues that are enriched in immune cells. These protocols are tailored to the cellular and tissue matrix composition of specific tissues in recognition of the fact that lung or intestinal tissues differ from lymphoid organ tissues. In order to process multiple samples from a single donor, we organize teams of individuals that work simultaneously using tissue site-specific protocols for tissues derived from each donor. Thus, for processing of tissue from an individual organ donor, mucosal, lymphoid, and blood protocols are all performed simultaneously, optimally by a team of 8±2 individuals. Processing can also be accomplished by fewer individuals, but in this case it will take longer. We have found that the most effective means of completing these protocols in a timely manner (so as to ensure cell viability) is to have all participants, regardless of their initial protocol designation, complete tasks on an “as needed” basis. This requires continual communication between all participants and substantial attention to proper sample labeling.

While these protocols are written in a linear fashion (and are characterized by individual steps), tissues progress through the various protocols at a pace that is independent of the individuals participating in donor tissue processing; thus, processing of differing tissues does not necessarily proceed in parallel. In any case, to ensure the continual flow of tissue through the protocols it is necessary to prepare all media and to stockpile many of the needed consumables in advance. With these steps in place our lab is able to consistently process the following amounts of tissue within a 6–7hr work day: Spleen (5g), Blood (70mL), Bone Marrow (30mL), Lung (20g), Lung Lymph Nodes, Bronchioalveolar lavage fluid (20mL), Iliac Lymph nodes, Thymus (10g, if available), Tonsils (if available), Jejunum (10g, Epithelial Layer and Lamina propria), Mesenteric Lymph Nodes, Ascending Colon (10g, Epithelial Layer and Lamina propria), Descending Colon (10g, Epithelial Layer and Lamina propria), Colonic Lymph Nodes, Liver (30g), and Hepatic Lymph Nodes. Effective execution of these protocols requires knowledge of the availability of protocol participants and adjustment of collection/processing goals based on this availability. Tissue processing teams are notified 12–24hrs in advance regarding donor availability. Related to this is the expectation that individuals participating in tissue processing be available to process tissue throughout the work week and willingness to schedule/reschedule other experiments to accommodate the availability of donor tissue. Finally, an on-call schedule ensures that donor tissue received over the weekend is processed by at least 3 individuals.

Basic Protocol 1: Isolation of immune cells from blood rich sites (Spleen [SPL], Blood [BLD], Bone Marrow [BOM])

Blood and Bone Marrow specimens obtained from donation source are received in anti-coagulant collection tubes; Spleen is received in UW (University of Wisconsin) media. Following plasma and serum collection, blood and bone marrow samples are diluted and immune cells are isolated by density centrifugation. The spleen is mechanically dissociated prior to immune cell isolation by density centrifugation.

Samples:

• Blood is retained in anti-coagulant collection tubes at room temperature on rotator until processing.

• Bone marrow is retained in anti-coagulant collection tubes at 4°C until processing.

• Spleen is retained in UW media at 4°C until processing.

Solutions:

• Iscove’s Modified Dulbecco’s Medium (IMDM) (Fisher Scientific, Cat. No.: 12-440-053)

• Complete Media (see Reagents and Solutions)

• Complete Buffer (see Reagents and Solutions)

Consumables and Supplies:

• Greiner Bio-One VACUETTE^™ Z Serum Sep Clot Activator Tubes (Fisher Scientific, Cat. No. 22-040-546) *Optional

• 100μM cell strainer (Fisher Scientific, Cat. No.: 50-146-1428)

• 50mL Centrifuge Tube (Fisher Scientific, Cat. No.: 12-565-271)

• Pestle (Fisher Scientific, Cat. No.: 50-197-8234)

• Ficoll-Paque^™ PLUS Media (Fisher Scientific, Cat. No.: 45-001-749)

• Falcon^™ Plastic Disposable Transfer Pipets (Fisher Scientific, Cat. No.: 13-680-50)

• Cryostor CS10 (Fisher Scientific, Cat. No.: NC9930384)

• Cryogenic Vials (Fisher Scientific, Cat. No.: 09-761-71)

• Mr. Frosty^™ (Fisher Scientific, Cat. No.: 51000001)

  1. Plasma Collection: Centrifuge whole blood (10 minutes, 400g at 20°C) in anti-coagulant tubes, remove plasma layer and store in cryovials (2mL/vial). Replace the plasma volume taken with Complete Buffer.
  2. Optional Serum Collection: Aliquot 1mL of whole blood into a Serum Sep Clot Activator Tube and centrifuge 1200g at 20°C for 10 minutes.
  3. Determine total blood volume and total bone marrow volume (record for determination of yield).
  4. Dilute blood and bone marrow with 4 volumes of Complete Buffer. Then dilute to bring volume to the nearest 25mL (e.g., 63mL of blood + 252mL (63mLx4) of Complete Buffer + additional 10mL of Complete Buffer = 325mL of diluted blood). Reserve diluted blood and bone marrow at room temperature until step 15.
  5. Remove capsule from spleen using forceps and a scalpel.
  6. Partition spleen to be used (we generally partition 5grams). Record weight of partitioned spleen (for determination of yield).
  7. Finely mince spleen in 50mL conical tube with 5mL of Complete Buffer. After mincing is complete bring volume up to ~50mL
     It is generally easiest to mince 0.5–1 gram at a time to get the most consistent small pieces.
  8. Disperse tissue across 100μM cell strainers resting in 50mL conical tubes, ~0.25g of tissue per filter.
  9. Mechanically dissociate the tissue over the cell strainer using a pestle. Periodically pour small amounts of Complete Buffer over the tissue until tissue has passed through the filter, leaving behind only residual connective tissue.
  10. With Complete Buffer, fill tubes up to 50mL then centrifuge (10 minutes, 400g at 20°C).
  11. Decant liquid fraction from centrifuged tubes then carefully recombine spleen fractions into a single 50mL conical tube using Complete Buffer.
  12. Bring the spleen suspension up to 50mL with Complete Buffer then filter the suspension through a 100μM cell strainer into a new tube.
  13. Evenly distribute the filtered spleen suspension across 50 mL conical tubes such that there is ~2 grams of tissue (round recorded spleen weight up to the nearest gram) per conical tube. If an odd number of grams of tissue was collected distribute 1 gram worth of suspension into a tube by itself (e.g. 4.8 grams spleen is rounded to 5 grams; thus, the 50mL of spleen suspension would be split across three 50mL conical tubes; 20mL in two of the 50mL conical tubes and 10mL in the third 50mL conical tube).
  14. Tubes with 2 grams of spleen suspension are brought up to a volume of 50mL using Complete Buffer. Tubes with 1 gram of spleen suspension are brought up to 25mL of volume using Complete Buffer.
  15. Determine the number of Ficoll tubes needed based on the volumes of tissue (25mL of diluted tissue [blood/bone marrow/spleen] per Ficoll tube).
  16. Label 50mL conical tubes with appropriate tissue name and add 15mL of Ficoll to the tube (e.g., 5 grams spleen is spread across 125mL of diluted cell suspension. This requires 5 Ficoll tubes).
  17. Carefully layer 25mL of cell suspensions on top of the Ficoll in an appropriately labeled tube. Final volume should now be 40mL per tube (15mL Ficoll + 25mL cell suspension).
  18. Centrifuge layered suspensions (20 minutes, 1200g 20°C with 4/9 acceleration and 0/9 brake [i.e. brake off]). Make sure the centrifuge is balanced and loaded as evenly as possible across all 4 swing buckets.
  19. Carefully remove mononuclear cell layer from each conical tube using a transfer pipet and combine into a new 50mL conical. Be careful to not mix cells from different tissues.
  20. Dilute cells with at least 1 additional volume of Complete Buffer. (e.g., if ~40mL are collected distribute this across two 50mL conical tubes and bring each up to 50mL with Complete Buffer).
  21. Centrifuge (10 minutes, 400g at 20°C) cell suspensions then decant liquid fraction.
  22. Combine cells into a single 50mL conical for each tissue and bring the volume up to 50mL with Complete Buffer. Be careful to not mix cells from different tissues.
  23. Centrifuge (10 minutes, 120g at 20°C) cell suspensions and decant liquid fraction to remove platelets.
  24. Resuspend cells in Complete Media.
  25. Count cells to determine both the cell concentration and, using the volume suspended, the total number of cells acquired from the tissue.
  26. Optional: Aliquot any cells needed for immediate use.
  27. Centrifuge (10 minutes, 400g at 20°C) cell suspensions and decant liquid fraction.
  28. Resuspend cells in Cryostor CS10 at a concentration of 2–3 ×10⁷ cells/mL.
  29. Aliquot Cryostor cell suspensions into Cryovials (1mL per tube).
  30. Label Cryovials with relevant information (e.g., Donor number, Tissue, Cell concentration, Date) then place in Mr. Frosty^™. Mr. Frosty^™ is placed in −80°C Freezer overnight. Vials are then moved to long-term storage in liquid nitrogen.

Basic Protocol 2: Isolation of immune cells from lymph nodes, tonsils, and thymus (Iliac Lymph Nodes [ILN], Lung lymph nodes [LLN], Mesenteric Lymph Nodes [MLN], Colonic Lymph Nodes [CLN], Hepatic Lymph Nodes [HLN], Tonsils [TON], Thymus [THY])

Tissues are received in UW media from donation source. Lymph nodes must be carefully extracted from tissue and/or surrounding adipose tissue and placed into complete media (Fig 2A). All tissues from these sites are treated identically and are collectively referred to as “lymphoid tissues”. Lymphoid tissues are finely minced prior to enzymatic liberation of cells from the surrounding matrix. Tissue is mechanically dissociated following enzymatic digestion. Immune cells are then isolated by density centrifugation.

Figure 2:

Visualizing key steps in tissue processing. A) Lymph nodes in the lung (LLN; left), small intestine associated mesentery (MLN; middle), and liver (HLN; right).

B) Acquiring bronchioalveolar lavage fluid (Basic protocol 3) step 4 (left) and step 5 (right).

C) Preparation steps for the Jejunum (Basic protocol 4) tissue enbloc step 1 (top left), step 3(top middle), step 4 (top right), step 5 (bottom left), step 7 (bottom middle), step 12 (bottom right).

Samples:

• Lymph nodes in tissues (see Basic Protocols 3–5) are retained in tissue in UW at 4°C until processing.

• Tonsils are retained in UW media at 4°C until processing.

• Thymus is retained in UW media at 4°C until processing.

Solutions:

• Iscove’s Modified Dulbecco’s Medium (IMDM) (Fisher Scientific, Cat. No.: 12-440-053)

• Complete Media (see Reagents and Solutions)

• Complete Buffer (see Reagents and Solutions)

Consumables and Supplies:

• Collagenase D (2.5g) (Sigma, Cat. No.: 11088882001)

• DNase (100mg) (Fisher Scientific, Cat. No.: NC9709009)

• EDTA 0.5M pH 8.0 (Fisher Scientific, Cat. No.: 15-575-020)

• 100μM cell strainer (Fisher Scientific, Cat. No.: 50-146-1428)

• 50mL Centrifuge Tube (Fisher Scientific, Cat. No.: 12-565-271)

• Pestle (Fisher Scientific, Cat. No.: 50-197-8234)

• Ficoll-Paque^™ PLUS Media (Fisher Scientific, Cat. No.: 45-001-749)

• Falcon^™ Plastic Disposable Transfer Pipets (Fisher Scientific, Cat. No.: 13-680-50)

• Cryostor CS10 (Fisher Scientific, Cat. No.: NC9930384)

• Cryogenic Vials (Fisher Scientific, Cat. No.: 09-761-71)

• Mr. Frosty^™ (Fisher Scientific, Cat. No.: 51000001)

  1. Determine the weight of each of the lymphoid tissues collected and record weights.
  2. Finely mince lymphoid tissues in 50mL conical tubes with 5mL of IMDM (no additives). After mincing is complete bring volume up to 40mL per gram of tissue.
     It is easiest to mince 0.5–1 gram at a time to get the most consistent small pieces. It is exceptionally important to very finely mince lymph nodes in order to effectuate robust cell extraction.
  3. Add 0.4mL of DNAse (0.1g/mL) and Collagenase (0.1g/mL) per 40mL of tissue suspension.
  4. Incubate tissue suspension on shaker/rotator for 30 minutes at 37°C.
  5. Add 0.5mL of EDTA per 40mL of tissue suspension and incubate at room temperature for 2 minutes.
  6. Disperse each tissue across 100μM cell strainers resting in 50mL conical tubes; ~0.25g of tissue per filter. Be careful to keep tissues separate.
  7. Mechanically dissociate the tissue over the cell strainer using a pestle. Periodically pour small amounts of Complete Buffer over the tissue until tissue has passed through the filter, leaving behind only residual connective tissue.
  8. With Complete Buffer, fill tubes up to 50mL then centrifuge (10 minutes, 400g at 20°C).
  9. Decant liquid fraction from centrifuged tubes then carefully recombine tissue fractions into a single 50mL conical for each tissue using Complete Media (red color helps to delineate Ficoll layers later). Be careful to keep tissues separate.
  10. Bring the tissue suspensions up to 50mL (or 25mL if less than 2 grams of tissue was acquired) with Complete Media then filter the suspensions through a 100μM cell strainer into a new tube.
  11. Evenly distribute the filtered cell suspensions across 50 mL conical tubes such that there is ~2 grams of tissue (round recorded tissue weight up to the nearest gram, if less than a gram progress as though it is a gram of tissue) per conical tube. If an odd number of grams of tissue was collected distribute 1 gram worth of suspension into a tube by itself (e.g., 0.6 grams iliac lymph node is rounded up to 1 gram thus this would stay in a single tube). Be careful to keep different tissues separated.
  12. Tubes with 2 grams of cell suspension are brought up to a volume of 50mL using Complete Media. Tubes with 1 gram or less of cell suspension are brought up to 25mL of volume using Complete Media.
  13. Determine the number of Ficoll tubes needed based on the volumes of tissue (25mL of diluted tissue per Ficoll tube).
  14. Label 50mL conical tubes with appropriate tissue name and add 15mL of Ficoll to the tube (e.g., 3 grams lung lymph node is spread across 75mL of diluted cell suspension. This requires 3 Ficoll tubes).
  15. Carefully layer 25mL of cell suspensions on top of the Ficoll in an appropriately labeled tube. Final volume should now be 40mL per tube (15mL Ficoll + 25mL cell suspension).
  16. Centrifuge layered suspensions (20 minutes, 1200g 20°C with 4/9 acceleration and 0/9 brake [i.e. brake off]). Make sure the centrifuge is balanced and loaded as evenly as possible across all 4 swing buckets.
  17. Carefully remove mononuclear cell layer from each conical tube using a transfer pipet and combine into a new 50mL conical tube. Be careful to not mix cells from different tissues.
  18. Dilute cells with at least 1 additional volume of Complete Buffer. (e.g., if ~40mL are collected distribute this across two 50mL conical tubes and bring each up to 50mL with Complete Buffer).
  19. Centrifuge (10 minutes, 400g at 20°C) cell suspensions then decant liquid fraction.
  20. Combine cells into a single 50mL conical for each tissue and bring the volume up to 50mL with Complete Buffer. Be careful to not mix cells from different tissues.
  21. Centrifuge (10 minutes, 400g at 20°C) cell suspensions and decant liquid fraction.
  22. Refer to Basic Protocol 1 steps 24–30 to complete protocol.

Basic Protocol 3: Isolation of immune cells from the lungs (Lung [LNG], Bronchioalveolar lavage [BAL])

Lung is received in UW media from donation source. A bronchioalveolar lavage is collected followed by isolation of lymph nodes (see Basic Protocol 2). Bronchioloalveolar lavage is then enzymatically digested to liberate cells and immune cells are isolated by density centrifugation. Lung tissue is finely minced followed by enzymatic digestion and subsequent mechanical dissociation. Lung immune cells are isolated by density centrifugation.

Samples:

• Lungs are retained in UW media at 4°C until processing.

Solutions:

• Dulbecco’s Phosphate Buffered Saline (DPBS) (Fisher Scientific, Cat. No.: 14-190-144)

• Iscove’s Modified Dulbecco’s Medium (IMDM) (Fisher Scientific, Cat. No.: 12-440-053)

• Complete Media (see Reagents and Solutions)

• Complete Buffer (see Reagents and Solutions)

Consumables and Supplies:

• BD Angiocath 10G (BD, Cat. No.: 382277)

• 3-Way Stopcock (Bio-Rad Laboratories, Cat. No.: 732-8103)

• 60mL Syringe (Fisher Scientific, Cat. No.: 14-955-461)

• 10mL Syringe (Fisher Scientific, Cat. No.: 14-955-459)

• Benzonase Nuclease (Millipore Sigma, Cat. No.: E1014-5KU)

• Collagenase D (2.5g) (Sigma, Cat. No.: 11088882001)

• DNase (100mg) (Fisher Scientific, Cat. No.: NC9709009)

• EDTA 0.5M pH 8.0 (Fisher Scientific, Cat. No.: 15-575-020)

• 100μM cell strainer (Fisher Scientific, Cat. No.: 50-146-1428)

• 50mL Centrifuge Tube (Fisher Scientific, Cat. No.: 12-565-271)

• Pestle (Fisher Scientific, Cat. No.: 50-197-8234)

• Ficoll-Paque^™ PLUS Media (Fisher Scientific, Cat. No.: 45-001-749)

• Falcon^™ Plastic Disposable Transfer Pipets (Fisher Scientific, Cat. No.: 13-680-50)

• Cryostor CS10 (Fisher Scientific, Cat. No.: NC9930384)

• Cryogenic Vials (Fisher Scientific, Cat. No.: 09-761-71)

• Mr. Frosty^™ (Fisher Scientific, Cat. No.: 51000001)

  1. Identify secondary bronchi that connect to the lower lobes of the lung and make an incision to allow insertion of a catheter into the bronchi.
  2. Insert a catheter (5–10cm in length) into a bronchus, remove attached needle (if present) and attach a 3-way stopper to the catheter. (Fig 2B, left)
  3. Fill a 60mL syringe with DPBS and connect it to the 3-way Stopper.
  4. Slowly inject 30mL of DPBS. Be sure to not overinflate the lung.
  5. Attach a 10mL syringe to remaining 3-way stopper port and collect 10mL of bronchioalveolar lavage fluid (BALF) from the lungs. Repeat until 25–50mL of BALF is collected (Fig 2B, right). Record volume collected.
     Repeat this procedure with other catheterized bronchi for additional BALF collection.
  6. Isolate bronchus- and lung-associated lymph nodes (to be used as LLN in Basic Protocol 2) and reserve lung tissue (~20grams) for processing below.
  7. Centrifuge (10 minutes 400g at 20°C) BALF and store supernatant (2mL per cryovial).
  8. Resuspend BAL cell pellet in 50mL of IMDM (no additives) and then add 0.05mL of benzonase to the BAL suspension and incubate for 30 minutes at 37°C.
  9. Add 0.5mL of EDTA to BAL suspension and then filter it through 100μM cell strainers. Proceed with BAL at step 22.
  10. Determine and record the weight of the lung tissue collected.
      Select an area separate from the BAL wash region.
  11. Finely mince lung tissue in 50mL conical tubes with 5mL of IMDM (no additives). After mincing is complete bring volume up to 40mL per gram of tissue.
      Depending on the amount of tissue this is best done in a single large volume container.
      It is easiest to mince 0.5–1 gram at a time in 5 mL to get the most consistent small pieces.
  12. Add 0.4mL each of DNAse (0.1g/mL) and Collagenase (0.1g/mL) per 40mL of tissue suspension.
  13. Incubate lung tissue suspension on shaker/rotator for 30 minutes at 37°C.
  14. Add 0.5mL of EDTA per 40mL of tissue suspension and incubate at room temperature for 2 minutes.
  15. Disperse lung tissue across 100μM cell strainers resting in 50mL conical tubes; ~0.25g of tissue per filter. Be careful to keep tissues separate.
  16. Mechanically dissociate the lung tissue over the cell strainer using pestle. Periodically pour small amounts of Complete Buffer over the tissue until tissue has passed through the filter, leaving behind only residual connective tissue.
  17. With Complete Buffer, fill tubes up to 50mL then centrifuge (10 minutes, 400g at 20°C).
  18. Decant liquid fraction from centrifuged tubes then carefully recombine tissue fractions into a single 50mL conical tube for each tissue using Complete Media (red color helps to delineate Ficoll layers later).
  19. Bring the lung cell suspension up to 50mL (or 25mL if less than 2 grams of tissue was acquired) with Complete Media. Distribute suspension across 50mL conical tubes so that there is no more than 5 grams of tissue per 50mL conical (e.g., if processing 20 grams of lung spread the 50mL across 5 tubes) this avoids overburdening the filter. Bring each tube up to 50mL with Complete Media then filter the suspensions through a 100μM cell strainers into new tubes.
  20. Evenly distribute the filtered lung cell suspensions across 50 mL conical tubes such that there is ~2 grams of tissue (round recorded tissue weight up to the nearest gram) per conical tube. If an odd number of grams of tissue was collected distribute 1 gram worth of suspension into a tube by itself (e.g. 18.7 grams lung is rounded up to 19 grams thus this would be separated into 9 conical tubes with 2 grams and 1 conical tube with 1 gram).
  21. Tubes with 2 grams of cell suspension are brought up to a volume of 50mL using Complete Media. Tubes with 1 gram or less of cell suspension are brought up to 25mL of volume using Complete Media.
  22. Determine the number of Ficoll tubes needed based on the volumes of tissue (25mL of diluted tissue per Ficoll tube). Be sure to include 2 tubes for the BAL.
  23. Refer to Basic Protocol 2 steps 14–22 to complete protocol.

Basic Protocol 4: Isolation of immune cells from the intestines (Jejunum Epithelial Layer [JEL], Jejunum Lamina Propria [JLP], Colon Epithelial Layer [CEL], Colon Lamina Propria [CLP])

Intestines are received in UW media from donation source. Surgeons demarcate regions of the small intestine prior to lab receiving tissue. Mesenteric and Colonic adipose tissue is removed and lymph nodes collected (see Basic Protocol 2). Intestinal sections are then taken and fecal content is removed. ‘Cleaned’ sections then undergo 2 rounds of epithelial stripping to differentiate cells in the epithelial and laminate propria layers of the section. Lamina propria layers are finely minced, enzymatically digested, and mechanically dissociated. Epithelial and lamina propria fractions then undergo a second enzymatic digestion prior to density centrifugation for immune cell isolation.

Samples:

• Intestine and associated mesentery/adipose tissue is retained en bloc in UW media at 4°C until processing.

Solutions:

• Iscove’s Modified Dulbecco’s Medium (IMDM) (Fisher Scientific, Cat. No.: 12-440-053)

• Gut Wash Buffer (see Reagents and Solutions)

• DTT Media (see Reagents and Solutions)

• Complete Media (see Reagents and Solutions)

• Complete Buffer (see Reagents and Solutions)

Consumables and Supplies:

• 250mL Conical Centrifuge Tube (Fisher Scientific, Cat. No.: 12-566-441)

• Collagenase D (2.5g) (Sigma, Cat. No.: 11088882001)

• DNase (100mg) (Fisher Scientific, Cat. No.: NC9709009)

• EDTA 0.5M pH 8.0 (Fisher Scientific, Cat. No.: 15-575-020)

• 100μM cell strainer (Fisher Scientific, Cat. No.: 50-146-1428)

• 50mL Centrifuge Tube (Fisher Scientific, Cat. No.: 12-565-271)

• Pestle (Fisher Scientific, Cat. No.: 50-197-8234)

• Benzonase Nuclease (Millipore Sigma, Cat. No.: E1014-5KU)

• Ficoll-Paque^™ PLUS Media (Fisher Scientific, Cat. No.: 45-001-749)

• Falcon^™ Plastic Disposable Transfer Pipets (Fisher Scientific, Cat. No.: 13-680-50)

• Cryostor CS10 (Fisher Scientific, Cat. No.: NC9930384)

• Cryogenic Vials (Fisher Scientific, Cat. No.: 09-761-71)

• Mr. Frosty^™ (Fisher Scientific, Cat. No.: 51000001)

  1. Remove mesentery using surgical scissors. Mesenteric lymph nodes (MLN) are then extracted from the mesentery to be used in Basic Protocol 2. (Fig 2C, top left)
  2. Remove Colon associated adipose tissue using surgical scissors. Colonic lymph nodes (CLN) are then extracted from the colonic adipose tissue to be used in Basic Protocol 2.
  3. Gently massage as much fecal content as possible out of the regions to be taken for tissue processing. (Fig 2C, top middle)
  4. Double ligate both ends of regions of the small and large intestine to be used for tissue processing. For example, ligate a portion of the jejunum [JEJ], ascending colon [aCOL], and descending colon [dCOL]. (Fig 2C, top right). Using surgical scissors cut between ligated regions. (Fig 2C, bottom left)
     Ligation ensures that fecal content from tissue not being processed does not flow into processed areas.
  5. Extrude any additional intestinal content from pieces to be used. This intestinal content can either be preserved for future analyses (e.g., microbiome analysis) or extruded directly into waste containers with bleach.
  6. Using surgical scissors, cut along the length of intestinal tissue such that the tissue is splayed open as a single flat piece. (Fig 2C, bottom middle)
  7. Trim tissue to the approximate amount of tissue desired for processing (generally we process 10 grams per intestinal region but wash additional tissue).
  8. Transfer intestinal pieces into 250mL conical containing ~100mL Gut Wash Buffer.
  9. Gently swirl the tissue in the tube to remove additional intestinal content.
  10. Remove tissue and transfer to a new 250mL conical tube containing fresh Gut Wash Buffer.
  11. Repeat steps 10 and 11 until tissue is clear of intestinal content (3–5 times). (Fig 2C, bottom right)
      It may not be possible to remove all fecal debris at this step, but over washing the tissue can prematurely strip the epithelial layer and lose cells for later collection. It is important to try to balance the cleaning of the tissue with the possibility of stripping the epithelial layer. Therefore, the focus should be to remove the majority rather than all of the fecal material.
  12. Determine the weight of each of the intestinal sites collected and record weights.
  13. Place intestinal regions into individual bottles containing DTT Media (~250mL of media for 10 grams of tissue). Be careful to keep tissues separated.
  14. Agitate the tissue on shaker for 30 minutes at 37°C.
  15. Remove tissue from bottle and place in a second bottle of DTT Media. Reserve the previous bottle of DTT Media as the epithelial fraction for the tissue (e.g., DTT Media from the jejunum is reserved as the jejunum epithelial layer [JEL])
  16. Agitate the tissue in the new DTT Media bottle on the shaker for an additional 30 minutes at 37°C.
  17. Remove tissue from DTT Media (reserve the DTT media as additional epithelial fraction for corresponding tissue) and place into a 250mL conical tube containing 100mL of Gut Wash Buffer.
  18. Using forceps agitate the tissue in the Gut Wash Buffer.
  19. Transfer the tissue to a new 250mL conical tube containing fresh Gut Wash Buffer. Combine the previous Gut Wash Buffer wash with the appropriate epithelial layers in DTT. Be careful to keep tissues separate.
  20. Repeat steps 19 and 20 an additional 3–4 times to thoroughly rinse the epithelial layer from the tissue.
  21. Transfer the tissue to a 50mL conical tube containing IMDM (no additives) and reserve until step 31.
  22. Combine Epithelial layers in DTT Media and Complete buffer for each tissue. Be careful to keep tissues separate. Label fractions as _EL as corresponds to the tissue site (e.g., ascending colon may be labeled as aCEL)
  23. Filter _EL fractions through 100μM cell strainers into 50mL conical tubes.
  24. Centrifuge (10 minutes, 400g at 20°C) _EL fractions and decant liquid fraction.
  25. Combine cell pellets for each tissue site into a single tube. Be careful to keep tissues separated.
  26. Bring the volume of each _EL fraction up to 50mL with IMDM (no additives).
  27. Centrifuge (10 minutes, 400g at 20°C) _EL fractions and decant liquid fraction.
      This is to remove any extraneous DTT Media.
  28. Resuspend the _EL fractions in 50mL of IMDM (no additives) then distribute each fraction across two 50mL conical tubes and bring each of these up to 50mL with IMDM (no additives). There should now be two 50mL conical tubes for each _EL fraction. Be careful to keep tissues separate.
  29. Proceed with _EL fractions at step 40.
  30. Finely mince each intestinal site from step 22 in 50mL conical tubes with 5mL of IMDM (no additives). After mincing is complete bring volume up to 40mL per gram of tissue.
      It is generally easiest to mince 0.5–1 gram at a time to get the most consistent small pieces.
  31. Add 0.4mL each of DNAse (0.1g/mL) and Collagenase (0.1g/mL) per 40mL of tissue suspension.
  32. Incubate tissue suspension on shaker/rotator for 30 minutes at 37°C. Label tissues as _LP as corresponds to the tissue site (e.g., descending colon may be labeled as dCLP)
  33. Add 0.5mL of EDTA per 40mL of tissue suspension and incubate at room temperature for 2 minutes.
  34. Disperse each tissue across 100μM cell strainers resting in 50mL conical tubes; ~0.25g of tissue per filter. Be careful to keep tissues separate.
  35. Mechanically dissociate the tissue over the cell strainer using pestle. Periodically pour small amounts of Complete Buffer over the tissue until tissue has passed through the filter, leaving behind only residual connective tissue.
  36. Dilute tubes up to 50mL then centrifuge (10 minutes 400g at 20°C).
  37. Decant liquid fraction from centrifuged tubes then carefully recombine tissue fractions into a single 50mL conical for each tissue using IMDM (no additives). Be careful to keep tissues separate.
  38. Bring the _LP fractions up to 50mL using IMDM (no additives) then distribute each fraction across two 50mL conical tubes and bring each of these up to 50mL with IMDM (no additives). There should now be two 50mL conical tubes for each _LP fraction. Be careful to keep tissues separate.
  39. Add 0.05mL of benzonase to each 50mL conical of _EL and _LP fractions. There should be two conical tubes for each _EL and _LP fraction for a total of four conical tubes for each intestinal site.
  40. Incubate _EL and _LP fractions at 37°C for 30 minutes.
  41. Add 0.25mL of EDTA to each _EL and _LP conical then rest for 2 minutes.
  42. Filter _EL and _LP fractions through 100μM cell strainers into fresh 50mL conical tubes.
  43. Divide each filtered fraction across five 50mL conical tubes (i.e., ~20mL of filtered fraction in each tube). This should lead to five conical tubes per _EL fraction and five conical tubes per _LP fraction for a total of ten conical per intestinal site. Be careful to keep tissues separate.
  44. Bring the _EL and _LP fractions up to 50mL with Complete Media.
  45. Determine the number of Ficoll tubes needed based on the volumes of tissue (25mL of diluted tissue per Ficoll tube; 10 Ficoll tubes are needed for each _EL and each _LP fraction).
  46. Refer to Basic Protocol 2 steps 14–22 to complete protocol.

Basic Protocol 5: Isolation of immune cells from the liver (Liver [LVR])

Liver is received in UW media from donation source. Hepatic lymph nodes are isolated (see Basic Protocol 2). Liver tissue is sectioned then finely minced, enzymatically digested, and mechanically dissociated. Liver immune cells are then isolated by density centrifugation.

Samples:

• Liver is retained in UW media at 4°C until processing.

Solutions:

• Iscove’s Modified Dulbecco’s Medium (IMDM) (Fisher Scientific, Cat. No.: 12-440-053)

• Complete Media (see Reagents and Solutions)

• Complete Buffer (see Reagents and Solutions)

Consumables and Supplies:

• Collagenase D (2.5g) (Sigma, Cat. No.: 11088882001)

• DNase (100mg) (Fisher Scientific, Cat. No.: NC9709009)

• EDTA 0.5M pH 8.0 (Fisher Scientific, Cat. No.: 15-575-020)

• 100μM cell strainer (Fisher Scientific, Cat. No.: 50-146-1428)

• 50mL Centrifuge Tube (Fisher Scientific, Cat. No.: 12-565-271)

• Pestle (Fisher Scientific, Cat. No.: 50-197-8234)

• Percoll PLUS (GE Healthcare, Cat. No.: 17-5445-01)

• Cryostor CS10 (Fisher Scientific, Cat. No.: NC9930384)

• Cryogenic Vials (Fisher Scientific, Cat. No.: 09-761-71)

• Mr. Frosty^™ (Fisher Scientific, Cat. No.: 51000001)

  1. Isolate hepatic lymph nodes (for basic protocol 2) then section the liver for processing, removing capsule.
  2. Determine the weight of the liver tissue collected and record weight.
  3. Finely mince liver in 50mL conical tubes with 5mL of IMDM (no additives). After mincing is complete bring volume up to 40mL per 2 grams of tissue.
     It is easiest to mince 0.5–1 gram at a time to get the most consistent small pieces.
  4. Add 0.4mL each of DNAse (0.1g/mL) and Collagenase (0.1g/mL) per 40mL of tissue suspension.
  5. Incubate tissue suspension on shaker/rotator for 30 minutes at 37°C.
  6. Add 0.5mL of EDTA per 40mL of tissue suspension and incubate at room temperature for 2 minutes.
  7. Disperse each tissue across 100μM cell strainers resting in 50mL conical tubes; ~0.25g of tissue per filter. Be careful to keep tissues separate.
  8. Mechanically dissociate the tissue over the cell strainer using pestle. Periodically pour small amounts of Complete Buffer over the tissue until tissue has passed through the filter, leaving behind only residual connective tissue.
  9. Dilute tubes up to 50mL then centrifuge (10 minutes, 400g at 20°C).
  10. Decant liquid fraction from centrifuged tubes then carefully recombine tissue fractions into a single 50mL conical for each tissue using Complete Media. Be careful to keep tissues separate.
  11. Bring the liver cell suspension up to 50mL (or 25mL if less than 2 grams of tissue was acquired) with Complete Media. Distribute suspension across 50mL conical tubes so that there is no more than 5 grams of tissue per 50mL conical (e.g., if processing 20 grams of liver spread the 50mL across 5 tubes) this avoids overburdening the filter. Bring each tube up to 50mL with Complete Media then filter the suspensions through a 100μM cell strainers into a new 50mL conical tubes.
  12. Evenly distribute the filtered cell suspensions across 50 mL conical tubes such that there is ~2 grams of tissue (round recorded tissue weight up to the nearest gram) per conical. If an odd number of grams of tissue was collected distribute it evenly across the other tubes (e.g., if processing 20.9 grams of tissue, then evenly distribute the cell suspension across 10 tubes).
  13. Bring up all conical tubes with cell suspension to a volume of 30mL with Complete Media.
  14. Add 10mL of Percoll to each tube to create a 25% Percoll solution.
  15. Gently invert tubes to mix.
  16. Centrifuge Percoll cell suspensions (20 minutes, 1200g 20°C with 4/9 acceleration and 0/9 brake [i.e. brake off]). Make sure the centrifuge is balanced and loaded as evenly as possible across all 4 swing buckets.
  17. Carefully suction off fat layer at the top and the excess Percoll media volume (leaving ~5mL of residual volume ensures cell pellet is minimally disrupted.
  18. Combine cell pellets into a single volume
  19. Dilute cells with at least 3 additional volumes of Complete Buffer. This is needed to thoroughly dilute out the Percoll (e.g., if ~40mL are collected distribute this across four 50mL conical tubes and bring each up to 50mL with Complete Buffer).
  20. Centrifuge (10 minutes 400g at 20°C) cell suspensions then decant liquid fraction.
  21. Refer to Basic Protocol 2 steps 20–22 to complete protocol.

Basic Protocol 6: Immune cell staining for flow cytometry

Procedures for the immunofluorescent antibody labeling of immune cells (specifically T cells) either immediately following isolation from tissue or from cryorecovery of frozen cell aliquots. Stained samples are analyzed by spectral flow cytometers.

Samples:

• Freshly isolated tissue immune cells are kept in Complete Media at 4°C and are ready for immediate use.

• Cryopreserved cells are retained in liquid nitrogen tanks until ready to be thawed for interrogation.

Solutions:

• Iscove’s Modified Dulbecco’s Medium (IMDM) (Fisher Scientific, Cat. No.: 12-440-053)

• Complete Media (see Reagents and Solutions)

• Complete Buffer (see Reagents and Solutions)

Consumables and Supplies:

• Collagenase D (2.5g) (Sigma, Cat. No.: 11088882001)

• DNase (100mg) (Fisher Scientific, Cat. No.: NC9709009)

• 100μM cell strainer (Fisher Scientific, Cat. No.: 50-146-1428)

• 50mL Centrifuge Tube (Fisher Scientific, Cat. No.: 12-565-271)

• 15mL Centrifuge Tube (Fisher Scientific, Cat. No.: 12-565-271)

• Fluorescently labeled antibodies and multimers can be acquired from various vendors depending on specific staining objectives (Biolegend, BD Biosciences, ThermoFisher Scientific, Cytek, Sony Biotechnology, Immudex, NIH tetramer core) See Table 1 for example.

• BD Horizon^™ Brilliant Stain Buffer (BD, Cat. No. 563794)

• BD Cytofix^™ Fixation Solution (BD, Cat. No. 554655)

• BD Cytofix/Cytoperm^™ Fixation and Permeabilization Kit (BD, Cat. No. 554717)

• FoxP3/Transcription Factor Staining Buffer Kit (Tonbo, Cat. No. TNB-0607)

• 5mL Falcon^™ Round-Bottom Polypropylene Tubes (Fisher Scientific, Cat. No.: 14-959-11A)

Table 1:

Example of T cell Flow cytometry Panel

		Marker	Clone	Stain Type	Reference
Ultraviolet	BUV395	CD103	Ber-act8	Surface	BD Biosciences
	BUV496	CD3	SK1	Surface	BD Biosciences
	BUV737	PD1	EH12.1	Surface	BD Biosciences
	BUV805	CD8	RPA-T8	Surface	BD Biosciences
Violet	BV421	IL-2	MQ1–17H12	Intracellular	BioLegend
	Pacific Blue	Perforin	B-D48	Intracellular	BioLegend
	BV570	CD45RA	HI100	Surface	BioLegend
	BV650	CD4	SK3	Surface	BD Biosciences
	BV711	CD69	FN50	Surface	BioLegend
	BV750	CXCR5	RF8B2	Surface	BD Biosciences
Blue	BB515	CCR7	2-L1-A	Surface	BD Biosciences
	PerCP	CD14	63D3	Surface	BioLegend
		CD16	3G8	Surface	BioLegend
		CD19	HIB19	Surface	BioLegend
	BB700	IFNγ	B27	Intracellular	BD Biosciences
Yellow / Green	PE	Antigen Specific Dextramer	D21–1351	Surface	BD Biosciences
	PE-Dazzle594	IL-17A	BL168	Surface	BioLegend
	PE-Cy5	CD107a	H4A3	Surface	BD Biosciences
	PE-Cy7	TNFα	MAb11	Intracellular	BD Biosciences
Red	AF647	IL-21	3A3-N2	Intracellular	BioLegend
	AF700	CD45	2D1 / G043H7	Surface	BioLegend
	Zombie NIR	L/D	/	Live_Dead	BioLegend

If using Fresh cells proceed to step 13; if using frozen cells proceed from step 1.

• 1Collect samples to be stained from liquid nitrogen storage and keep frozen on either dry ice or in a −80°C freezer.
• 2Pre-warm IMDM (no additives) and add 5mL each of DNAse (0.1g/mL) and Collagenase (0.1g/mL) per 500mL bottle.
• 3Add 5mL of IMDM with DNAse and Collagenase to 15mL conical tubes (have a labeled 15mL conical for each sample you intend to thaw).
• 4Thaw samples (no more than 2 at a time) in a 37°C water bath.
• 5When sample is almost completely thawed add ~1mL of warmed IMDM with DNAse and Collagenase then transfer drop wise to labeled 15mL conical tube while agitating. Rinse cryovial twice then set it aside.
• 6Repeat steps 4–5 until all samples are thawed.
• 7Centrifuge (10 minutes 400g at 20°C) cell suspensions then decant liquid fraction.
• 8Add 5mL of IMDM with DNAse and Collagenase to 15mL conical tubes.
• 9Allow cells to rest for 15min at room temperature.
• 10Filter cell suspension through 100 μM filter into fresh labeled 15mL conical tubes.
• 11Centrifuge (10 minutes 400g at 20°C) cell suspensions then decant liquid fraction.
• 12Resuspend cells in 1mL of Complete Media.
• 13Count cells to determine the cell concentration.
• 14Plate 2–5 ×10⁶ cells per well in a 96-well round bottom plate. Also plate compensation beads in separate wells as needed.
• 15Centrifuge (10 minutes 400g at 20°C) cell suspensions then decant liquid fraction.
• 16Resuspend cells in 200μL Complete Buffer.
• 17Repeat steps 15–16 twice.

  Optional: If staining with antigen specific multimers (tetramers or dextramers) complete steps 18–20. Otherwise add 50mL of Complete Buffer and proceed to step 21.

• 18Make a master mix of multimers along with FC block (typically 1μL each of each dextramer and the FC block per 50μL of Complete buffer).
• 19Add 50mL of multimer master mix to each sample (be sure to include appropriate multimer control stains).
• 20Incubate plate at room temperature in the dark for 30 min.
• 21Make a 2X concentration master mix of your surface stain using Complete Buffer and antibodies for surface staining (can include viability dye with surface antibody stain). Include BD Horizon^™ Brilliant Stain Buffer if using several Brilliant Violet or Brilliant Ultraviolet antibodies. Incorporate additional FMO master mixes as needed depending on the surface markers being stained.
• 22Add 50μL of surface stain master mix to cells for staining.
• 23Incubate plate at 4°C for 30 min.
• 24Centrifuge (10 minutes 400g at 20°C) cell suspensions then decant liquid fraction.
• 25Resuspend cells in 200μL Complete Buffer.
• 26Repeat steps 24–25 twice.
• 27Samples can either be run immediately or fixed using fixation buffer. If running cells immediately resuspend the cells in 100μL of Complete Buffer and run on flow cytometer. Examples of multimer and general flow cytometry staining can be observed in (Gordon et al., 2017). If fixing cells but not performing intracellular stain proceed to steps 28–32. If fixing cells and performing intracellular staining (excluding transcription factors) proceed to steps 33–40. If fixing cells and performing intracellular staining that includes transcription factors proceed to steps 41–48.

Fixation no intracellular stain:

• 28Add 50μL of Cytofix^™ and incubate plate at 4°C for 30 min.
• 29Centrifuge (10 minutes 400g at 20°C) cell suspensions then decant liquid fraction.
• 30Resuspend cells in 200μL Complete Buffer.
• 31Repeat steps 29–30 twice.
• 32Resuspend cells in 100uL of Complete buffer and run on flow cytometer.

Fixation/Permeabilization with intracellular stain (no transcription factors):

• 33Add 100μL of Cytofix/Cytoperm^™ then incubate plate at 4°C for 30 min.
• 34Centrifuge (10 minutes 600g at 20°C) cell suspensions then decant liquid fraction.
• 35Make a master mix of intracellular antibodies using Permeabilization Wash Buffer.
• 36Stain cells with 100μL intracellular antibody master mix 4°C for 1hr.
• 37Centrifuge (10 minutes 600g at 20°C) cell suspensions then decant liquid fraction.
• 38Resuspend cells in 200μL Permeabilization Wash Buffer.
• 39Repeat steps 38–39 twice.
• 40Resuspend the cells in 100μL of Complete Buffer and run on flow cytometer.

Fixation/Permeabilization with intracellular stain (with transcription factors):

• 41Add 100μL of FoxP3 Transcription Factor Fixation/Permeabilization Buffer then incubate plate at 4°C for 2hrs to overnight.
• 42Centrifuge (10 minutes 600g at 20°C) cell suspensions then decant liquid fraction.
• 43Make a master mix of intracellular antibodies using Permeablization Wash Buffer.
• 44Stain cells with 100μL intracellular antibody master mix 4°C for 2hrs to overnight.
• 45Centrifuge (10 minutes 600g at 20°C) cell suspensions then decant liquid fraction.
• 46Resuspend cells in 200μL Permeabilization Wash Buffer.
• 47Repeat steps 45–46 twice.
• 48Resuspend the cells in 100μL of Complete Buffer and run on flow cytometer.

Reagents and Solutions

All solutions should be prepared and used in biosafety cabinets to promote sterility. Prepared solutions should be stored at 4°C.

Complete media – IMDM (500mL), Penicillin-Streptomycin-Glutamine (5mL), FBS (50mL)

• Iscove’s Modified Dulbecco’s Medium (IMDM) (Fisher Scientific, Cat. No.: 12-440-053)

• Penicillin-Streptomycin-Glutamine (100X) (Fisher Scientific, Cat. No.: 10-378-016)

• Fetal Bovine Serum (FBS) (Fisher Scientific, Cat. No.: 10-099-14)

Gut Wash Buffer – DPBS (500mL), FBS (25mL)

• Dulbecco’s Phosphate Buffered Saline (DPBS) (Fisher Scientific, Cat. No.: 14-190-144)

• Fetal Bovine Serum (FBS) (Fisher Scientific, Cat. No.: 10-099-14)

DTT Media – IMDM (500mL), Penicillin-Streptomycin-Glutamine (5mL), FBS (50mL), EDTA (10mL), DTT (1mL; final concentration 2mM)

• Iscove’s Modified Dulbecco’s Medium (IMDM) (Fisher Scientific, Cat. No.: 12-440-053)

• Penicillin-Streptomycin-Glutamine (100X) (Fisher Scientific, Cat. No.: 10-378-016)

• Fetal Bovine Serum (FBS) (Fisher Scientific, Cat. No.: 10-099-14)

• EDTA 0.5M pH 8.0 (Fisher Scientific, Cat. No.: 15-575-020)

• DTT (Cell Signaling Technology, Cat. No.: #7016)

Complete Buffer - DPBS (500mL), FBS (25mL), EDTA (2mL)

• Dulbecco’s Phosphate Buffered Saline (DPBS) (Fisher Scientific, Cat. No.: 14-190-144)

• Fetal Bovine Serum (FBS) (Fisher Scientific, Cat. No.: 10-099-14)

• EDTA 0.5M pH 8.0 (Fisher Scientific, Cat. No.: 15-575-020)

Commentary

Understanding Results

Our access to a large quantity and variety of human tissue has provided a unique opportunity to study T lymphocytes and other immune cells in various parts of the body and to identify tissue-specific variations while internally controlling for high variability of the human population. With this access we interrogate the influence of tissue on the phenotype and function of immune cells located in tissues, particularly T_RM cells.

Our studies have allowed us to establish expectations of yield for each of the tissues processed. Despite the fact that humans are highly heterogeneous, we observe reproducible trends in immune cell yield across our donor cohort. For example, the average yield of cells from lymph nodes is highest for the LLNs and MLNs (Fig 3A), but the reason these two tissue sources provide high yields differ. The LLNs consist of large nodes that line the trachea whereas the MLNs consist of numerous small lymph nodes embedded in the mesenteric adipose tissue. While lymph nodes generally have good viability (Fig 3B), the smaller lymph nodes (i.e., MLNs) exhibit variations in cellular content per gram of tissue based on age; donors>50yrs have fewer numbers of MLNs, reduced immune cell content, and increased fibrosis (Lam et al., 2024) (Fig 3C). Thus, the overall high yield from LLNs and MLNs arises from the fact that the number of cells harvested from the relatively few high yielding LLNs is balanced by the number of cells from the relatively many low yielding MLNs. Colonic and hepatic lymph nodes by contrast tend to be large but less frequent than their mesenteric counterparts and thus have a per gram cell yield more similar to the LLNs.

Figure 3:

Tissue Yield and Viability Expectations. A) Grams of indicated lymph nodes collected per donor.

(B) Percentage of live cells recovered for indicated tissue per donor.

(C) Number of viable cells recovered per mL [BLD,BOM] or gram [all others] of tissue per donor. (D) Frequency of T cells per tissue representative of each protocol.

Box and Whisker indicate mean with 95% confidence interval. Tissue Abbreviations: BLD - Blood, BOM - Bone Marrow, SPL - Spleen, ILN - Iliac Lymph Nodes (LNs), CLN - Colonic LNs, LLN - Lung LNs, HLN - Hepatic LNs, MLN - Mesenteric LNs, BAL - Bronchioalveolar Lavage, LNG - Lung, JEL - Jejunum Epithelial Layer (EL), JLP - Jejunum Lamina Propria (LP), IEL - Illeum EL, ILP - Illeum LP, aCEL - ascending Colonic EL, aCLP – ascending Colonic LP, dCEL - descending Colonic EL, dCLP – descending Colonic LP, LVR - Liver. Color Coding: Red - Protocol 1, Yellow - Protocol 2, Blue - Protocol 3, Green - Protocol 4, Orange - Protocol 5. Data (A-C) are pooled from 185 unique donors ranging from 19–185 donors per tissue. Data (D) are pooled from 5–46 donors.

It is apparent that the methods necessary for extraction of cells from tissues impacts their viability with Protocols 3–5 tending toward lower viability. Further, the removal of stromal cells, which are depleted in the density gradients, likely contributes to the lower cell yield per gram of tissue. Related to this is the trend toward reduced viability of cells from the epithelial layers relative to the lamina propria of the corresponding gut site. We find that this lower viability is mostly due to the presence of epithelial cells that may still be present in the epithelial layer fractions.

It should still be noted that heterogeneity across donors is to be expected and that it is common for individual tissues to have an abnormally high or low yield in a given donor. As such we would caution against over-interpreting or extrapolating based on an individual donor or even a small sampling of donors.

To best express the abundance of T cells in tissues we have included the frequency of total T cells among live CD45⁺ cells present in tissue derived from of each of the protocols outlined and then quantified by flow cytometry (Fig 3D). The latter is performed using optimized high dimensional spectral flow cytometry panels, such as the one present in Table 1. These panels allow us to interrogate T cell subsets, antigen specificities, phenotypic characteristics, function, and transcription factor utilization. When utilizing spectral flow cytometry to assess tissue lymphocytes it is important to appropriately titrate each panel and stain an equivalent number of cells from each tissue. Furthermore, using appropriate unstained cells helps establish donor-specific variations in autofluorescence. These steps ensure the most reproducible data and are critical in limiting donor-to-donor variability.

We have performed several single cell transcriptomic analyses using cells obtained directly after isolation from tissues using the above protocols (Gray et al., 2024; Matsumoto et al., 2023; Poon et al., 2023; Wells et al., 2024). In addition, we performed immunofluorescent imaging and more recently spatial transcriptomics using the tissues as prepared using the above protocols (Matsumoto et al., 2023; Ural et al., 2022; Verma et al., 2024). In both cases, this has led to high quality data.

Critical Parameters and Troubleshooting

While our experience with these protocols has revealed general trends and expected yields for tissue there is still significant donor heterogeneity and poor yields can be due to that specific donor tissue rather than an error in the processing of that tissue. However, there are several factors that can influence yield which a researcher should be cognizant of while executing these protocols. Many of the aspects of tissue quality come down to timing: the time between death and medical intervention, the time elapsing from when the tissue is removed from the body to when it is actually processed, and the time it takes to get the cells out of the tissue once the tissue processing has begun. Further, areas of edema, restricted circulation, or tissue fibrosis can impact the cell yield as well as the donor’s age and medical history. Therefore, it is important to review any available donor information and visually inspect the region being taken prior to processing.

It is also important to consider the available labor needed to perform the tissue processing. This includes both the number of individuals available on a given day to process the tissue as well as the degree of experience of those available. As stated, the time it takes to process the tissue available can impact the cell yield; thus, attempting to process too much tissue and therefore taking excessive time to process tissue can negatively impact yield and viability. This becomes particularly relevant in view of the fact that this aspect of tissue quality is the factor that researchers tend to have the most control and potential to improve yields.

In view of the above, it can be important to consider how many cells each stakeholder (researcher needing cells) requires for their experiment and the relative importance of a given donor for each stakeholder when considering how much of each tissue to process. For example, it is exceptionally easy to take additional spleen which has a high cell yield per gram of tissue whereas lymph nodes are limiting and the labor cost on sites like intestinal tissue can be tremendous if a lot of tissue is required. These labor and limiting tissue factors are especially relevant in the context of the effort needed to complete all five of these protocols for single donor that, as mentioned, takes 8 people processing tissue for 6–7 hours to complete on average.

When considering modifications to these protocols it is important to control the manipulations within a given donor because there can be a significant degree of donor-to-donor variability and without the internal comparison the results are likely to be uninterpretable. With this in mind, testing any potential modifications comes with added labor or yield cost wherein considerations of how much tissue to process can influence stakeholder expectations for a given donor. This includes what may appear to be relatively minor changes to the protocol, including attempts to reduce labor by using specialized equipment. For example, our protocol uses scissors to manually chop tissue prior to enzymatic digestion. While there are several machines available for purchase that may accomplish this same task, in our attempts to utilize them we have noted impacts on yield, with biased impacts on specific cell populations (e.g., specific loss of B cells and macrophages), that has made them incompatible for our use. While it is possible and even probable that there is some setting that may allow for the use of these machines with our tissue, the labor and yield costs necessary to elucidate the exact parameters needed for each tissue were deemed too high. Therefore, a manual approach has served to be the most consistent. Further, any advance in one step may necessitate advances in other steps to make the most effective use of whatever change is implemented. To use the prior example, a machine that enables chopping more tissue subsequently requires more labor in tissue homogenization, layering cell suspensions on Ficoll, removing the cell layers post-Ficoll, and progressing tissue through the various stages of the protocol. So, even small changes require assessment of downstream impact. By contrast, an example of an easy implementation to reduce effort was the inclusion of a dispenser for adding a consistent volume of Ficoll to tubes prior to sample layering. This reduced the labor/time in filling tubes without necessitating other changes to the protocol. This may seem a minor improvement, however, when considering the >100 Ficoll tubes that are filled to complete these protocols per donor even modest labor reductions can yield substantive results.

For troubleshooting areas of weakness in our protocols we take a proactive approach of consistent retraining on the different protocols. This is because, while all individuals are expected to help with all protocols, people still tend to fall into a routine of which tissues they process, particularly at the start of the day. To retain individual competency in all protocols, we routinely switch which protocol an individual may start on. This also ensures that no protocol is entirely dependent on one or a few individuals’ knowledge base. If a generalized issue is being seen across multiple individuals, it can be worthwhile to discuss how everyone is performing the protocols to ensure there is not deviation and potentially check reagents if the problem is systemic. More specific examples of issues are raised in the following ‘Troubleshooting Table’. Ultimately, however, the best way to identify issues with a particular donor tissue is by performing analysis (in our case this generally consists of performing flow cytometry to assess the cellular composition of the sample) on the sample relative to prior samples collected. This should give an indication if the sample is truly an outlier possibly due to tissue processing, or if an abnormality in yield is merely due to human heterogeneity.

Troubleshooting Table

Problem	Possible Cause	Solutions
Individuals loss of familiarity with aspects of protocols	Ingrained routine	Rotate individual on to deficit protocol Keep all protocols posted in tissue processing area for reference
Exceptionally low viability (<20%)	Poor tissue quality or too long to process	Ensure regions of best visual appearance are used Limit time to process where available Reduce quantity of tissue processed as needed
Poor yield post-Ficoll	Poor Ficoll layering or collection	Retraining as needed Ensure cell suspensions are filtered prior to layering EDTA not added to suspension prior to layering Ensure cell suspension is layered properly (limited mixing) Check centrifuge speed/acceleration/break
Poor viability and yield generally	Cells not properly extracted	Ensure tissue is finely chopped pre-enzymatic digestion (especially LNs) Check enzymes are not old/expired Ensure that individuals are completely homogenizing digested tissue through filter post-digestion
Loose cell pellets	Too many red blood cells/platelets (Protocol 1) Too many epithelial cells (Protocol 4) Too much Percoll (Protocol 5)	Lyse red blood cells with ACK lysis buffer if needed Ensure Platelet spin was done Decant as normal don’t try to preserve loose epithelia, epithelial cells will be loose (fluffy) and decanting rather than trying to preserve them will improve yield/viability (should not take lymphocytes with them) Ensure appropriate mix of Percoll and media

Time Considerations:

Prepping solutions and consumables prior to donor processing generally takes 2 individuals a half an hour. This is done as soon as the surgeons have accepted an organ donor case. The tissue processing itself generally takes a team of 8 people approximately 6 hours to complete assuming all 5 of the tissue processing protocols are being performed. This is somewhat shorter if the intestinal site is unavailable as Protocol 4 tends to be the most labor intensive and time consuming of the protocols. Staining of samples can take anywhere from 2 to 18 hours depending on the complexity of the staining being undertaken (multimers and intracellular stains will extend time required and transcription factor staining involves an overnight fixation). Additionally, if using frozen samples appropriate time should be budgeted for sample thawing and cleanup prior to staining (thawing more samples will require more time and delay when staining can commence).

Data Availability

Data will be provided upon reasonable request.