Protocol for collection and generation of bat primary lung and kidney cells

Summary

Some bat species host pathogenic viruses and represent recognized models for studying antiviral immunity. Here, we present a protocol for collecting and generating primary cells from wild bat tissues. We describe steps for tissue collection, enzymatic digestion, isolation, and cell expansion. We then detail procedures for immunocompetency testing of the established cultures. This protocol describes the generation of in vitro models for understudied non-model species, facilitating physiologically relevant assays to improve understanding of bat biology, immunology, and pathogen-host interactions.

Graphical abstract

Highlights

• •Guidelines for capturing and harvesting lungs and kidneys from wild bats
• •Tissue-specific approaches for establishing primary cell cultures
• •Guidance for confirming the species of primary cell cultures using PCR
• •Procedure to confirm immunocompetence of primary cell cultures

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Some bat species host pathogenic viruses and represent recognized models for studying antiviral immunity. Here, we present a protocol for collecting and generating primary cells from wild bat tissues. We describe steps for tissue collection, enzymatic digestion, isolation, and cell expansion. We then detail procedures for immunocompetency testing of the established cultures. This protocol describes the generation of in vitro models for understudied non-model species, facilitating physiologically relevant assays to improve understanding of bat biology, immunology, and pathogen-host interactions.

Before you begin

Bats represent the second most diverse mammalian order and account for over 1,500 species distributed across all continents except Antarctica.¹ Some bat species have been recognized as hosts for highly pathogenic zoonotic viruses, some of which are able to cause severe disease in humans and livestock.^2,3 However, bat species that are infected with these viruses often do not develop severe disease.^4,5,6 Work over the last three decades has explored various biological characteristics that enable bats to resist or tolerate viral infections. Yet the lack of bat-specific tools remains a major limitation in the field. Bat cell lines have been developed from only a very limited number of species, constraining the possibilities of obtaining physiologically relevant data. This is a significant limiting factor, because the immune response across bat species cannot be generalized and needs to be studied within specific host–virus settings. Therefore, increasing the taxonomic breadth and diversity of available bat in vitro models will significantly improve the quality of the data being produced, which will increase our overall understanding of bat biology and pathogen–host interactions.⁷

Here, we describe the process of tissue collection and enzymatic digestion using trypsin for bat kidneys and DNase I-pronase cocktail for bat lungs. Tissues were collected from two focal Neotropical bat species known to harbor potentially zoonotic viruses, including those in the Coronaviridae family: the little yellow-shouldered Mesoamerican bat (Sturnira parvidens) and the northern black mastiff bat (Molossus nigricans).^8,9 Both species have a conservation status of least concern, enabling limited lethal sampling (IUCN Red List of Threatened Species). Tissues were digested for several time periods to allow for appropriate isolation of single cells to be subsequently cultured and maintained until confluency and storage. In addition, we validated the application of these newly developed kidney- and lung-derived primary cell cultures by assessing their immunocompetence and suitability as models for virological studies. Immunocompetence was assessed through transfection with polyinosinic:polycytidylic acid [poly(I:C)], followed by infection with vesicular stomatitis virus engineered to express a green fluorescent protein (VSV-GFP). Poly(I:C) is a synthetic double-stranded RNA analog that mimics viral infection and stimulates an antiviral interferon response. VSV-GFP presents a safe reporter virus that enables rapid infection readout. Decreased VSV-GFP infection levels indicate that cells are able to mount a competent immune response to poly(I:C) stimulation.

Innovation

Bats are being increasingly recognized as hosts of multiple zoonotic viruses including filoviruses, coronaviruses, henipaviruses, and rabies virus, among others. Despite causing serious disease in other mammalian species, these viruses do not typically cause clinical signs of disease in experimentally or naturally infected bats. Thus, bats are an intriguing model to study host–virus interactions to discover antiviral processes that have evolved to specifically counteract these groups of viruses. Moreover, primary cells are critical tools for studying host–virus interactions. To date, few studies have reported the establishment of primary cell cultures from bat tissues. For example, Crameri et al. established primary cultures from multiple organs of Pteropus alecto using tissue dissociation and culture prior to immortalization.¹⁰ Likewise, Banerjee et al. attempted to establish primary cultures from lungs, kidneys, and other tissues from Eptesicus fuscus using trypsin-based dissociation; however, only kidney-derived cells were successfully isolated and expanded.¹¹ These studies demonstrated feasibility of generating bat cell cultures, but detailed tissue-specific protocols for generating and establishing primary bat cells remain limited, particularly for lung tissues (Table 1). Here, we build upon previous work by providing a detailed, step-by-step workflow for generating primary bat cells from both kidney and lung tissues. In addition to tissue processing and culture establishment, the protocol includes procedures for cell expansion, cryopreservation, species confirmation by cytochrome b sequencing, and functional assessment of antiviral immunocompetency.

Table 1.

Two available protocols for establishment of bat primary cell cultures

	Crameri et al.10	Banerjee et al.11	This manuscript
Species	Pteropus alecto	Eptesicus fuscus	Molossus nigricans, Sturnira parvidens
Storage	Ice, immediately processed after euthanasia	Immediately processed after tissue collection	Flash frozen, long-term
Tissues attempted	Aorta, bone marrow, brain, foetus, foetal membranes, heart, kidney, liver, lymph nodes, lungs, muscle, pharynx, placenta, salivary gland, small intestine, skin, spleen, testes, thymus, uterus	Brain, liver, lungs, spleen, kidney.	Lungs, kidney
Primary tissues established	All (as above)	Kidney	Lungs, kidney
Disassociation method	Overnight (16–18 h) 0.25% trypsin-EDTA at 4°C	0.5% trypsin-EDTA at room temperature (20°C–22°C). Time and number of digestions N/A	Kidney: 3x 30′ 0.25% trypsin-EDTA at room temperature (20°C–22°C) Lungs: overnight (16–18 h) pronase and DNase I at 4°C followed by RBC lysis at room temperature (20°C–22°C)
Species confirmation	g-banding, Apolipoprotein B 100 Precursor gene	cytochrome b	cytochrome b

Institutional permissions

Prior to sampling wild bats, researchers must obtain permission from relevant institutions, including but not limited to animal care and use committees, wildlife agencies, and other governmental entities. For this study, field sampling was approved by the Institutional Animal Care and Use Committee of the University of Oklahoma (protocol R21-021), the Belize Forestry Department (scientific collection permits FD/WL/1/24 (06) and FD/WL/1/22(07)), and the Belize Institute of Archaeology (permit IA/H/1/24(02)). Tissue samples were imported into the United States through approval from the Belize Forestry Department (permits FD/WL/7/24(56) and FD/WL/7/24(57)) and the Centers for Disease Control and Prevention (permit 20240306-0860A). Tissues were imported into Canada through approval from the Canadian Food Inspection Agency. We also note the importance of assessing species conservation status (e.g., via the IUCN Red List of Threatened Species or local conservation prioritization) when deciding on the need for lethal sampling as well as sample size. All laboratory studies were conducted under containment level 2 conditions. Researchers should consult with their institutional biosafety office prior to starting work.

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Bacterial and virus strains
Vesicular Stomatitis Virus-Green Fluorescent Protein	Dr. Karen Mossman	N/A
Biological samples
Kidney	This study (Molossus nigricans and Sturnira parvidens, male individuals, wild caught adult bats with specific age unknown)	N/A
Lung	This study (as above)	N/A
Chemicals, peptides, and recombinant proteins
DMEM (Dulbecco’s Modified Eagle’s Medium)	Thermo Fisher Scientific	Cat# 11965126
DMEM/F-12	Thermo Fisher Scientific	Cat# 11320033
MEM (Minimum Essential Medium)	Thermo Fisher Scientific	Cat# 11-095-098
Fetal Bovine Serum (FBS)	Cytiva	Cat# SH30396.03
PBS, pH 7.4	Thermo Fisher Scientific	Cat# 10010031
Trypsin-EDTA (0.5%)	Thermo Fisher Scientific	Cat# 15400054
GlutaMAX supplement	Thermo Fisher Scientific	Cat# 35050061
L-Glutamine (200 mM) (100X)	Thermo Fisher Scientific	Cat# 25030081
Penicillin-streptomycin	Thermo Fisher Scientific	Cat# 15140122
Antibiotic-antimycotic (100X)	Thermo Fisher Scientific	Cat# 15240062
MEM Non-Essential Amino Acids Solution (100X)	Thermo Fisher Scientific	Cat# 11140050
HEPES (1M)	Thermo Fisher Scientific	Cat# 15630106
Dimethyl sulfoxide (DMSO)	Sigma-Aldrich	Cat# D2650
Micro-Chem Plus	National Chemical Laboratories, Inc.	N/A
NaCl	Sigma-Aldrich	Cat# S9625
Lipofectamine 3000 Transfection Reagent	Sigma-Aldrich	Cat# L3000015
OptiMEM	Sigma-Aldrich	Cat# 31985062
Poly(I:C) (HMW) - TLR3 Agonist	Invivogen	Cat# tlrl-pic
2-Mercaptoethanol, 99%, pure	Thermo Fisher Scientific	Cat# 125472500
Oligonucleotides
Cytb – Forward Primer (5’ – CCC CHC CHC AYA TYA ARC CMG ART GAT A – 3′)	Parson et al.12	N/A
Cytb – Reverse Primer (5’ – TCR ACD GGN TGY CCT CCD ATT CAT GTT A – 3′)	Parson et al.12	N/A
Software and algorithms
Evos Analysis Software	Thermo Fisher Scientific	N/A
Other
Microscope (EVOS™ M7000 Imaging System)	Thermo Fisher Scientific	Cat# AMF7000
6-well cell culture plate	Corning Incorporated	Cat# 3516
12-well cell culture plate	Corning Incorporated	Cat# 3513
T100 Thermal Cycler	Bio-Rad	Cat# 1861096
Dual LED Blue/White Light Transilluminator	Thermo Fisher Scientific	Cat# LB0100
Corning® 15 mL centrifuge tubes	Sigma-Aldrich	Cat# CLS430055-100EA
Corning® 50 mL centrifuge tubes	Sigma-Aldrich	Cat# CLS430829-500EA
Stripettes	Sarstedt	Cat# 86.1254.001
Pipet-Aid XP	Drummond	Cat# 4000101
Centrifuge	Thermo Fisher Scientific	Sorvall ST40R Centrifuges
Amcor Parafilm™ M Laboratory Film	Thermo Fisher Scientific	Cat# 1337410
TC flask T25	Sarstedt	Cat# 83.3910.002
TC flask T75	Sarstedt	Cat# 83.3911.002
Mr. Frosty freezing container	Sigma-Aldrich	Cat# C1562-1EA
Sterile micro-dissecting scissors	Sigma-Aldrich	Cat# S3146
Sterile tweezers	Sigma-Aldrich	Cat # 930229
Pronase	Sigma-Aldrich	Cat# 10165921001
Deoxyribonuclease I from bovine pancreas	Sigma-Aldrich	Cat# D5025-15KU
Falcon® 70 μm Cell Strainer	Thermo Fisher Scientific	Cat# 08-771-2
Sterile Syringe Filter Unit, MCE, 0.22 μm	Thermo Fisher Scientific	Cat# SLGL0250S
BD General Use Syringes (30 mL)	Thermo Fisher Scientific	Cat# 22-124-969
eBioscience™ 1X RBC Lysis Buffer	Thermo Fisher Scientific	Cat# 00-4333-57
RNeasy Plus Mini Kit	Qiagen	Cat# 74134
PCR Using Q5® High-Fidelity DNA Polymerase	New England Biolabs	Cat #M0491
Agarose	Thermo Fisher Scientific	Cat# BP160100
iScript™ cDNA Synthesis Kit	Bio-Rad	Cat #1708891
Monarch® Spin DNA Gel Extraction Kit	New England Biolabs	Cat #T1120S/L

Materials and equipment

DMEM medium (kidney and lung epithelial cells)

Reagent	Final concentration	Amount
DMEM	N/A	430 mL
FBS	10%	50 mL
Antibiotic-antimycotic	2X	10 mL
GlutaMAX supplement	1X	5 mL
MEM Non-Essential Amino Acids Solution	1X	5 mL
Total	N/A	500 mL

Note: Medium may be prepared in advance and stored at +4°C no more than 6 months. Warm up to 37°C before using. For VSV-GFP propagation, use serum-free DMEM supplemented with 1% penicillin-streptomycin. Serum-free DMEM may be stored at 4°C for 3 months. Warm media in a 37°C water bath before use.

MEM digestion medium

Reagent	Final concentration	Amount
MEM	N/A	21 mL
Pronase (10 mg/mL)	1.4 mg/mL	3.5 mL
Deoxyribonuclease I from bovine pancreas (5 mg/mL)	0.1 mg/mL	500 μL
Total	N/A	25 mL

Note: Prepare enzymatic working solutions as per the manufacturer’s instructions and sterilize them through a 0.22 μm membrane prior to proceeding. We recommend preparing the digestion media immediately before proceeding, as enzymes lose activity when stored at +4°C.

NEC basal medium (lung fibroblasts)

Reagent	Final concentration	Amount
DMEM/F-12	N/A	447.5 mL
FBS	10%	25 mL
HEPES (1M)	25 mM	12.5 mL
Antibiotic-antimycotic	2X	10 mL
MEM Non-Essential Amino Acids Solution	1X	5 mL
Total	N/A	500 mL

Note: Medium may be prepared in advance and stored at +4°C no more than 6 months. Warm media in a 37°C water bath before use.

Step-by-step method details

Bat tissue collection and storage

Timing: 1–2 days

This step describes the procedures used to capture wild bats, perform humane euthanasia, collect relevant tissue samples, and store them properly for preservation.

Note: All laboratory studies were conducted under containment level 2 conditions. Researchers should consult with their institutional biosafety office prior to starting work.

• 1.
  Prepare collection reagents.

  • a.
    Thaw FBS and anti-anti (antibiotic-antimycotic) concentrate in water bath.
  • b.
    Under a biosafety cabinet (BSC), combine 90 mL FBS and 10 mL DMSO.
  • c.
    Under a BSC, discard 3 mL media and add 3 mL anti-anti.
  • d.
    Under a BSC, add 1 mL media to sterile cryogenic vials.
  • e.
    Store prepared media tubes at −20°C until tissue collection.
  • f.
    Under a BSC, add 1 mL DNA/RNA Shield to sterile cryogenic vials.
  • g.
    Store prepared DNA/RNA Shield tubes at room temperature (20°C−22°C) until tissue collection.
• 2.
  Capture wild bats.

  Note: Ensure proper PPE during capture and handling of bats, including minimally latex and leather gloves, KN95/N95 mask, pre-exposure prophylaxis for rabies virus and other lyssaviruses, and vaccination against SARS-CoV-2.

  Note: Geographic context and known zoonotic viruses circulating in focal bat species will ultimately determine further PPE (e.g., eye protection, Tyvek suits).

  • a.
    Use mist-nets, hand nets, or harp traps to humanely capture bats while foraging or upon leaving/returning to roost sites.
  • b.
    Safely extract bats from the nets or harp trap and temporarily hold them in individual cloth bags.
  • c.
    Use morphological keys to identify bats to species.
  • d.
    Determine age, sex, and reproductive status via morphology.
• 3.
  Collect tissue samples.

  • a.
    Use 1–5 mL isoflurane to induce anesthesia via inhalation.
  • b.
    Confirm anesthesia through toe pinch, dilated pupil, and absence of heartbeat
  • c.
    Perform euthanasia via thoracic compression or cervical dislocation.
  • d.
    Prior to tissue collection, additional PPE, such as eye protection (e.g., safety goggles, face shield), should be adopted.
  • e.
    Use a sterile scalpel to cut from the pubic bone to the throat, expose subcutaneous tissue, and pin skin to avoid contaminating internal organs.
  • f.
    Use sterile scissors to make an incision from the pubic bone up to the sternum, opening the abdominal cavity.
  • g.
    Collect the kidney and remove two 1–2 mm pieces of tissue. Wash tissue in sterile PBS in a sterile petri dish.
  • h.
    Store one piece of kidney tissue in the prepared media tube (see step 1), tightly close and rotate the cryogenic vial to ensure tissue is submerged entirely in media, and store immediately in liquid nitrogen.
  • i.
    Store the other piece of kidney tissue in the prepared DNA/RNA Shield tube (see step 1), tightly close and invert the cryogenic vial several times to ensure tissue is submerged entirely in buffer and store.
  • j.
    Use sterile scissors to cut open the diaphragm and gain access to the thoracic cavity.
  • k.
    Collect the lungs and remove two 1–2 mm pieces of tissue. Wash tissue in sterile PBS in a sterile petri dish.
  • l.
    Store one piece of lung tissue in the prepared media tube, tightly close and invert the cryogenic vial several times to ensure tissue is submerged entirely in media, and store immediately in liquid nitrogen.
  • m.
    Store the other piece of lung tissue in the prepared DNA/RNA Shield tube, tightly close and rotate the cryogenic vial to ensure tissue is submerged entirely in buffer and store.
  • n.
    Work with a museum to voucher the carcass and any remaining tissue samples if possible.
  • o.
    Clean and re-sterilize instruments with 10% bleach and 70% ethanol between individual bats.
• 4.
  Maintain cold chain until laboratory processing.

  • a.
    Following collection, maintain cryogenic vials with organ tissue in liquid nitrogen.
  • b.
    Refill liquid nitrogen in dewars or recharge liquid nitrogen dry shippers as needed.
  • c.
    Following collection, store paired tissue samples in DNA/RNA Shield at −80°C.

Note: A fully charged dry shipper provides an effective alternative to liquid nitrogen in resource-limited settings. Once fully charged, it can maintain the required cryogenic temperature for approximately 10–20 days, depending on its size and manufacturer, thereby ensuring the preservation of temperature-sensitive samples.

Note: Dry ice or a portable −80°C freezer (e.g., ULT25NEU, Stirling) may be used in similar resource-limited settings to store samples that have less stringent temperature storage requirements. However, such limitations also highlight the critical need to invest in research capacity, particularly for molecular analyses, in such regions.

Generation of kidney-derived primary cells

Timing: 3 h

This step details the procedure for generating primary cells from bat kidney tissue through sequential trypsin digestions.

Note: Trypsin is a pancreatic enzyme that hydrolyzes long peptide chains into smaller peptides, thereby allowing tissue dissociation into single primary cells for culture.

• 5.
  Recover the tissue sample from liquid nitrogen.

  • a.
    Thaw tube in 37°C water bath.
  • b.
    Disinfect the tube by spraying 70% ethanol.
  • c.
    Open the tube inside a clean BSC and place the contents inside one well of a 6-well plate.
• 6.
  Mince the kidney tissue.

  • a.
    Remove freezing media from the well using a serological pipette.
  • b.
    Wash tissue with 1–2 mL of room temperature (20°C–22°C) PBS in the well to remove excess blood and freezing media.
  • c.
    Add PBS over the tissue to prevent the tissue from drying out.
  • d.
    Carefully mince the tissue using sterile dissecting scissors.

Note: The plate may be slightly inclined to ensure all tissue pieces converge at the bottom. This will minimize any unwanted spread and consequent loss of small pieces of tissue throughout the well, therefore maximizing the amount of tissue collected in the digestion solution.

• 7.
  Start enzymatic digestion.

  • a.
    Prepare a 0.25% trypsin solution in a 50 mL tube by diluting 7.5 mL of 0.5% trypsin in 7.5 mL of room temperature (20°C–22°C) PBS.
  • b.
    Add 5 mL of 0.25% trypsin over the minced tissue using a serological pipette.
  • c.
    Transfer the contents of the well to a 15 mL centrifuge tube.
  • d.
    Ensure the tube is tightly closed by adding laboratory film around the cap.
  • e.
    Agitate the tissue on a tube roller for 30 min at room temperature (20°C–22°C).

Note: Since we used a high trypsin concentration, 30 mins at room temperature (20°C–22°C) is sufficient for each step. Long incubation times can affect cell viability.

• 8.
  Stop enzymatic digestion.

  • a.
    Let the tube stand in a tube rack for 5 min, allowing for the minced tissue to settle to the bottom of the tube.
  • b.
    Add 2 mL of FBS to a new 15 mL tube.

    Note: Higher volumes of FBS may be used if greater volumes or higher concentrations of trypsin are used for tissue digestion.

  • c.
    Transfer the supernatant of the digestion solution to the tube containing FBS. Leave remaining pieces of tissue in the original tube to proceed with further digestions (see step 10).
  • d.
    Close the tube containing FBS tightly and invert it 5 times to neutralize the digestion.
• 9.
  Resuspend recovered single cells in complete media.

  • a.
    Centrifuge tube containing FBS and supernatant for 5 min at 200 × g.
  • b.
    Carefully remove supernatant from the tube using a serological pipette, without disturbing the cell pellet.
  • c.
    Resuspend the cell pellet in 1 mL of complete DMEM medium.
  • d.
    Transfer cell suspension to a well of a 6-well plate, using a serological pipette.
  • e.
    Incubate cells at 37°C humidified incubator with 5% CO₂.
• 10.
  Repeat digestion two extra times for the undigested minced tissue.

  • a.
    To the tissue chunks (from step 8.c), add 4 mL of trypsin.
  • b.
    Repeat steps 7.b-9.e, adding the subsequent cell suspensions to the same well of the 6-well plate to obtain a final volume of 3 mL.
  • c.
    Fill the remaining empty wells in the plate with sterile PBS or complete media to prevent loss of media by evaporation.
• 11.Incubate cells at 37°C humidified incubator with 5% CO₂ for 24 h.
• 12.Disinfect material and discard remaining pieces of tissue using 5% Microchem solution.

Development of lung-derived primary cells

Timing: 10–18 h

This step details the procedure for generating primary cells from bat lung tissues through pronase and DNase I digestions.

• 13.Thaw and mince tissues as described in steps 5 and 6.
• 14.
  Incubate minced tissue in MEM digestion medium.

  • a.
    Collect minced tissue into 25 mL of MEM digestion medium in a 50 mL tube.
  • b.
    Close the tube lid tightly and place laboratory sealing film around the cap.
  • c.
    Agitate tissue in a tube roller at 4°C for 6–14 h in the dark.

Note: We recommend 4°C for collagenase to protect cell viability and maintain structural integrity of delicate lung tissue. Researchers may try 30 mins at RT and assess tissue digestion and cell viability. End digestion once the media starts to turn slightly cloudy due to released cells and tissue chunks start to disintegrate.

• 15.
  Stop enzymatic digestion.

  • a.
    Add 2.5 mL of FBS to the MEM digestion media, corresponding to 10% of the total volume.
• 16.
  Filter cell suspension to remove undigested fragments present in the sample.

  • a.
    Pass cell suspension through a 70 μm cell strainer and into a new 50 mL tube.
• 17.
  Lyse red blood cells that may be present in the solution.

  • a.
    Centrifuge dissociated cells at 200 × g for 10 min at 4°C.
  • b.
    Remove supernatant using a serological pipette.
  • c.
    Resuspend the cell pellet in 10 mL of filter-sterilized 1x RBC lysis buffer.

    Note: We did not use the RBC lysis step for kidney tissue since in our hands we have been consistently able to generate bat kidney cells without the RBC lysis step. Researchers are welcome to try the RBC lysis step for kidneys if it increases their ability to develop primary cell cultures.

  • d.
    Incubate at room temperature (20°C–22°C) for 6 min with occasional shaking.
• 18.
  Obtain fibroblast-like cell population.

  • a.
    Centrifuge cells at 200 × g for 10 min at 4°C.
  • b.
    Remove supernatant from the tube using a serological pipette.
  • c.
    Resuspend cell pellet in 3 mL of complete NEC basal medium.
  • d.
    Seed cell suspension in a well of a 6-well plate.
  • e.
    Incubate cells for 2 h at 37°C humidified incubator with 5% CO₂ to allow fibroblasts to attach.
• 19.
  Obtain epithelial-like cell population.

  • a.
    After incubation in step 18.e., collect supernatant with non-adherent cells and transfer it to a 15 mL tube.
  • b.
    Replace well with NEC basal medium to culture fibroblast-like populations.
  • c.
    Centrifuge obtained supernatant at 200 × g for 10 min at room temperature.
  • d.
    Remove supernatant and resuspend cell pellet in 3 mL complete DMEM medium.
  • e.
    Seed cells in a new well of the same plate to culture epithelial-like populations.
• 20.Incubate cells at 37°C humidified incubator with 5% CO₂ for 24 h.
• 21.Disinfect material and discard remaining pieces of tissue using 5% Microchem solution.
• 22.
  Change media on the cells 24 h post-cell isolation.

  • a.
    Monitor cells daily and change media every 2–3 days.
• 23.When cells achieve confluency, split into a T25 and expand appropriately until freezing.

Note: We recommend freezing the cells as soon as possible and avoiding prolonged passaging to prevent adverse effects on primary cell metabolism and growth.

Passaging and freezing primary cells

Timing: 1 h

This step describes the procedure for passing and freezing cells for storage and later use.

• 24.Fill up the Mr. Frosty freezing container with 2-propanol. It may be reused up to 5 times.
• 25.
  Upon reaching 80%–90% confluency in a T75 flask, passage the cells.

  • a.
    Remove media and wash flask with warm PBS.
  • b.
    Trypsinize cells using 0.25% trypsin for 5–7 min at 37°C.
  • c.
    Neutralize trypsinization using appropriate complete media.
  • d.
    Transfer solution to a 15 mL tube. A small portion of these cells can be reseeded into a flask or tissue culture plate to maintain the culture.
  • e.
    Centrifuge the 15 mL tube for 7 min at 100 × g.
• 26.Prepare freezing media with 90% warm complete media and 10% sterile DMSO.
• 27.Resuspend cell pellet in 5 mL of freezing media for each T75 flask.
• 28.Aliquot 1 mL of cells per cryovial and place inside Mr. Frosty container at −80°C.
• 29.The next day, recover cryovials and store them in a liquid nitrogen tank for long-term storage.

Thawing of frozen primary cells

Timing: 30 min

This step outlines the procedure for recovering cryopreserved primary cells for further experimental assays.

• 30.Obtain cryovial from liquid nitrogen and quickly thaw in a 37°C bath.
• 31.Spray the cryovial with 70% ethanol for surface disinfection.
• 32.Add 1 mL of the cell suspension to 9 mL of warm complete media in a 15 mL tube.
• 33.Centrifuge the 15 mL tube for 7 min at 100 × g.
• 34.Resuspend cell pellet in 6 mL of complete media for one T25 flask, or 12 mL of complete media for one T75 flask. Seed cells.
• 35.Change media the next day and/or expand as appropriate.

Species confirmation of cell cultures by cytochrome b sequencing

Timing: 2 days

This step describes the procedure for confirming species of newly developed primary cell cultures by cytochrome b (cytb) sequencing.

Note: The cytb gene is a highly conserved mitochondrial gene widely used for biological identification as it contains species-specific sequence information.

• 36.
  Prepare and collect cell lysates.

  • a.
    Seed 1.5 × 10⁵ cells per well in a 12-well plate and incubate overnight (16–18h) at 37°C and 5% CO₂.
  • b.
    Once cells reach confluency, collect cell lysate by adding 350 μL per well of RLT Plus buffer (from the RNeasy Plus Mini Kit) prepared with fresh 2-mercaptoethanol (100:1; v:v).
• 37.Extract RNA from the collected lysates, as outlined in the manufacturer’s protocol.
• 38.Synthesize complementary DNA (cDNA) from 400 ng of the extracted RNA, using the iScript Reverse Transcription Kit, as outlined in the manufacturer’s protocol.
• 39.Prepare a PCR reaction to amplify part of the cytb gene sequence for each of the primary cell cultures generated using Q5 High-Fidelity DNA Polymerase and the manufacturer’s protocol.
• 40.Perform the PCR using the following cycling conditions (Table 2):
• 41.Analyze PCR products on 1% agarose gel.
• 42.Extract the cytb bands from the gel under a transilluminator.
• 43.Extract DNA from the fragments using the Monarch® Spin DNA Gel Extraction Kit, following manufacturer’s protocol.
• 44.Sequence the obtained amplicons.
• 45.
  Analyze sequencing results.

  • a.
    Use NCBI BLASTn analysis of the amplicon sequences against the Chiroptera order to confirm genus and species identity.

Table 2.

PCR cycling conditions

Steps	Temperature	Time	Cycles
Initial Denaturation	94°C	3 min	1
Denaturation	94°C	30 sec	35
Annealing	55°C	30 sec	35
Extension	72°C	10 sec	35
Final extension	72°C	10 min	1
Hold	4°C	N/A	N/A

Testing antiviral competency of primary cells

Timing: 3 days

This step describes the procedure for testing whether the newly generated primary cells can mount an antiviral response. Our approach was based on transfecting cells with polyinosinic:polycytidylic acid [poly(I:C)], a synthetic analog of viral double-stranded RNA that triggers the innate immune response. When performed in combination with a reporter virus infection, like VSV-GFP, this assay serves as a functional readout of antiviral immunity in the newly developed primary cell cultures.

Note: This assessment can be performed by using any reporter virus. If this is not possible, other viruses might be used alongside appropriate assessment of viral readouts such as TCID50 or plaque assays. Additionally, quantitative polymerase chain reactions (qPCR) for antiviral genes (such as MX1, IFIT1, IFNB) can be another tool to assess the antiviral competency of the newly developed cells. A cell line known to respond to poly(I:C) can be included in this step to act as a technical positive control in the assay

• 46.
  Poly(I:C) stimulation.

  • a.
    Seed 1.5 × 10⁵ cells per well in 12-well plates and incubate overnight (16–18h) at 37°C and 5% CO₂.
  • b.
    Prepare transfection mix.

    • i.
      Dilute the stock poly(I:C) solution in Opti-MEM to obtain a 10 ng/mL working solution.
    • ii.
      Prepare the transfection mixes separately for each well in 1.5 mL microcentrifuge tubes.
    • iii.
      To the 1.5 mL microcentrifuge tube, add 250 μL of Opti-MEM and the desired quantity of poly(I:C).

      Note: It is recommended to use 10–100 ng/mL of poly(I:C) for this protocol. If cells do not respond to these concentrations, it may be necessary to increase them or change the duration of poly(I:C) transfection in cells prior to virus infection.

    • iv.
      Vortex the mix for 15 s.
    • v.
      Vortex the lipofectamine 3000 tube for 15–20 s.
    • vi.
      Add 4 μL to each tube of transfection mix.
    • vii.
      Vortex the final transfection mix for 15–20 s.
  • c.
    Incubate transfection mix at room temperature for 15 min.
  • d.
    Add transfection mix to each well, dropwise, using a 1 mL pipette.
  • e.
    Incubate cells at 37°C humidified incubator with 5% CO₂ for 6 h, before proceeding to virus infection.

    Note: The stimulation period may be increased in case cells do not respond to poly(I:C) stimulation for 6 h. Both concentration and stimulation times might be optimized and can be cell-type dependent. In addition, researchers may include additional controls for scientific rigor based on their research questions like mock transfected cells, lipofectamine only treated cells, and virus only infected cells.

• 47.
  VSV-GFP challenge.

  • a.
    Prepare a virus inoculum of multiplicity of infection 0.05 in serum-free media.

    Note: A viral replication curve may be needed to optimize the right multiplicity of infection for each culture.

  • b.
    Remove complete media from the wells of the tissue culture plate.
  • c.
    Add 500 μL of the virus inoculum to each well.
  • d.
    Incubate plates for 1 h at 37°C with intermittent rocking every 15 min.
  • e.
    Remove virus solution from the cells and wash 3x with pre-warmed PBS.
  • f.
    Add 1 mL of pre-warmed complete media back to each well.
  • g.
    Incubate cells at 37°C humidified incubator with 5% CO₂.
  • h.
    The next day, image the cells using a fluorescence microscope, such as the EVOS™ M7000 Imaging System and assess the level of infection per condition.
  • i.
    Quantify number of GFP-positive cells using EVOS Analysis Software. We recommend analyzing a minimum of five non-overlapping fields per well and performing at least three independent biological replicates per experimental condition.

    Note: For increased statistical rigor, we recommend including at least three independent biological replicates per condition. Multiple fields of view (minimum of five per well) should be analyzed to account for within-well variability. Alternative image analysis softwares, such as ImageJ, may be used for infection quantification.

Expected outcomes

Primary cells are a valuable resource for research as they provide relevant models for investigating molecular processes and pathogen-host interactions. For wildlife species like bats, the generation of primary cells can aid in the study of immune responses.^11,13 Here, we present a tested, detailed step-by-step protocol for generating kidney- and lung-derived cells from bats. We also provide methods for an assay to test cell immunocompetency through immune stimulation and antiviral assays.

The number of cells obtained from the established cultures will depend on the original size of each animal species (larger animals have more tissue material), as well as on the tissue dicing technique. Primary cells from newly processed tissues should start attaching and growing in the plates during the first week (Figure 1). As is the case with primary cells, maintaining them post passage 5–10 is not recommended to avoid cell death, senescence, or other unwanted non-relevant physiological responses. If immortalized cell lines are preferred, several methods exist.^11,14,15

Figure 1.

Representative timelines for the establishment of primary cell cultures from bat tissues

Bright-field images illustrate the progression of primary cell culture establishment from (A) Sturnira parvidens kidney-derived cells and (B) Molossus nigricans lung-derived cells.

(A) Images were acquired at Day 0 (initial plating following tissue processing), Day 4 (first adherent colony observed), Day 7 (expanding colonies), Day 12 (cells dispersed following first passage), and Day 17 (confluent culture prior to cryopreservation).

(B) Images were acquired at Day 0 (initial plating following tissue processing), Day 2 (first adherent colonies observed) and Day 15 (confluent culture prior to passaging). All images were taken at 10x magnification, except for the Day 4 image in panel A (acquired at 20x) and Day 0 image in panel B (acquired at 4x). Scale bars are variable and indicated in the images.

Through the sequencing of the cytochrome b gene from the newly developed primary cells, genus- or species-level identity can be confirmed if reference genomes or partial target sequences are available in sequence repositories like NCBI. Cytochrome b is widely used to confirm wildlife species, due to the extensive number of sequences available in public databases. Upon optimization of poly(I:C) transfection and VSV-GFP infection levels (or other appropriate readout), cells are expected to show immunocompetency in response to viral stimulation in a cell- and virus-dependent manner (Figure 2). In our case, both newly generated S. parvidens kidney cells and M. nigricans lung primary cells demonstrated immunocompetence in our assays, albeit at different concentrations of poly(I:C). S. parvidens kidney cells show null infection levels upon stimulation with as little as 25 ng/mL poly(I:C), whereas M. nigricans lung cells require a minimum of 100 ng/mL poly(I:C) to show a decrease in viral replication. These results highlight tissue- and species-dependent differences that affect the overall susceptibility and immunocompetency of the generated cells.

Figure 2.

Newly developed primary cell cultures can mount an antiviral response

Sturnira parvidens kidney-derived cells and Molossus nigricans lung-derived cells demonstrate immunocompetency upon poly(I:C) stimulation. Primary cells were transfected with 0–100 ng/mL poly(I:C) for 6 hrs followed by VSV-GFP infection (0.05 MOI).

(A and B) S. parvidens kidney-derived cells, (C and D) M. nigricans lung-derived cells.

(A and C) BF (top) and GFP (bottom) images were obtained 24 hpi at 10x magnification.

(B and D) Quantification of VSV-GFP infection expressed as percentage of infection relative to the mean of the number of infected cells in VSV-GFP+ 0 ng poly(I:C) control. For all quantifications, each data point represents one well in the culture plate (mean of 5 images per well; n = 4 wells per condition, except M. nigricans VSV-GFP + 100 ng/ml poly(IC), where n=3). Bars indicate mean ± SEM. Statistical significance was assessed using one-way ANOVA with Tukey’s multiple comparisons test (ns, not significant; ∗∗p < 0.01; ∗∗∗∗p < 0.0001). Scale bar: 100 μm.

Limitations

The generation of primary cells from bat tissues has been described in other publications, but a detailed protocol such as this does not exist. Our goal in creating this protocol is to support the efforts of new researchers in the field and to help boost mechanistic studies into bat biology. The biggest limitation of this protocol is access to healthy, viable tissues from bat species. The number of research colonies of bats has grown, but these remain rare and cover far less than 1% of bat species diversity. Proper tissue preservation can also be a limiting factor, due to poor accessibility of some field work sites with inability to carry the most efficient equipment.

Once tissues are in the laboratory, the necessary kits and materials used in the protocols are not expensive and widely accessible to most laboratories. However, even with processing times of 1–2 days, cells may take several weeks to grow and establish a cell population. Moreover, variable cell-dependent susceptibility to available immunocompetency screening tools may require further optimization. It is worth noting that researchers should be aware of species-specific variabilities, if any, in cell culture conditions, potential contamination by microbes and the limited lifespan of primary cells. The population doubling time (PDL) of the generated cell lines was not measured but may be desirable if the observed growth kinetics differ significantly between batches or individual samples. Some other limitations of the study include: (i) the protocol was validated in two bat species from a single geographic region; applicability to other bat families (e.g., Pteropodidae, Rhinolophidae) should be tested and optimized by researchers and, (ii) immunocompetency assay tests dsRNA-sensing arm of innate immunity (like TLR3/MDA5 pathways) and it is recommended that researchers identify and characterize other innate immune pathways as relevant for their studies.

Troubleshooting

Problem 1

Low yield of primary cells (related to steps 4–6 and 13).

Potential solution

Cell viability is at its highest shortly after tissue harvesting. If tissues need to be stored prior to processing, freeze in liquid nitrogen in individual vials fully filled with storage media to make sure that tissues are embedded in it and not floating on top of the freezing media. Upon thawing, quickly warm the vial in a 37^°C water bath and immediately start processing. Additionally, cutting the tissues into smaller pieces may increase the surface area and, therefore, the chances of primary cell isolation in the digestion media.

Problem 2

Contamination of primary cell culture (related to steps 2 and 3).

Potential solution

Take precautions during tissue collection in the field to ensure that clean techniques are used and tissues are not in contact with external materials like soil. Concentration of antibiotics in the media may be increased to address mild contamination, but this is not ideal. Since the goal of these cells is to use them in downstream molecular studies, any hint of contamination while establishing these cells should lead to decontamination of cells, followed by discarding them using institutional biohazard disposal processes. Fresh tissues will have to be used to start a new culture.

Problem 3

Primary cells are growing very slowly (related to steps 18 and 22).

Potential solution

Attached cells may be transferred from a 6-well plate to a 12-well plate, as they may prefer to be in closer proximity to each other. Concentration of FBS in the media may be increased to help. Ensure that media is changed regularly, preferably every 2–3 days.

Problem 4

Primary cells are not attaching to the plate (related to steps 9 and 18).

Potential solution

Ensure that the plate containing processed primary cells remains undisturbed for the first 2–3 days to allow for proper cell attachment. If the problem persists, spin the cells and resuspend in fresh media before reseeding into the same plate.

Problem 5

Digestion yields no primary cells (related to steps 7 and 14).

Potential solution

For generating primary cells from lung tissues, digestion times vary widely to accommodate the characteristics of each tissue. If the well seems empty after processing, try a shorter digestion time to optimize the isolation of primary cells from that species.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Arinjay Banerjee (arinjay.banerjee@usask.ca).

Technical contact

Technical questions on executing this protocol should be directed to and will be answered by the technical contact, Arinjay Banerjee (arinjay.banerjee@usask.ca; cell culture development and characterization) and Daniel J. Becker (danbeck@ou.edu; field sample and tissue collection).

Materials availability

Cells developed in this study will be available from the lead contact with a completed material transfer agreement and biological agent transfer form.

Data and code availability

This protocol does not generate any original code. Any additional information may be available from the lead contact upon request.

Acknowledgments

This study was supported by a New Frontiers in Research Fund (NFRF) Exploration grant (NFRFE-2023-00025) awarded to D.J.B. and A.B. R.M.Q.-T. is partially supported by a Canadian Institutes of Health Research (CIHR) funded Canopy training program in One Health and zoonosis and a University of Saskatchewan College of Graduate and Postdoctoral Studies Dean’s scholarship. D.J.B. was also supported by the Edward Mallinckrodt Jr. Foundation. A.B.R. was supported by the Human Frontier Science Program (LT0017/2024-L). D.J.B. and A.V.-S. were supported by the National Science Foundation (DBI 2515340). N.B.S. was supported by the Taxonomic Mammalogy Fund of the American Museum of Natural History. This work was undertaken, in part, thanks to funding from the Canada Research Chairs Program awarded to A.B. (CRC-2024-00291). VIDO receives operational funding from the Government of Saskatchewan through Innovation Saskatchewan and the Ministry of Agriculture and from the Canada Foundation for Innovation through the Major Science Initiatives Fund. The graphical abstract was generated using BioRender.com.

Author contributions

Conceptualization, R.M.Q.-T., M.M.T.P., and A.B.; methodology, R.M.Q.-T., M.M.T.P., A.V.-S., B.L.O., B.R.A., D.J.B., and A.B.; data interpretation, R.M.Q.-T. and M.M.T.P.; writing and editing, R.M.Q.-T., M.M.T.P., K.B., N.B.S., and A.B.; funding and resources, D.J.B., A.B., and N.B.S.; supervision, D.J.B. and A.B.

Declaration of interests

A.B. is an inventor of the Efk3B cell line.