Isolation and Culture of Human-Induced Pluripotent Stem Cell-Derived Cerebral Organoid Cells

Abstract

The advent of human-induced pluripotent stem cell (iPSC)-derived three-dimensional (3D) cerebral organoids provides unprecedented opportunities of modeling human brains in states of health and disorder. Emerging data supports that cerebral organoids allow for more relevant in vitro systems for studying the human brain system and diseases than the current widely used 2D monolayer cell culture. Thus, the ability to isolate, culture, and maintain human brain cells from cerebral organoids is highly needed, particularly for studies on organoid-derived cell-type-specific signaling and their electrophysiological properties. Here we present a protocol to isolate and culture brain cells from 2-month human iPSC-derived cerebral organoids. The dissociation and plating of cells from organoids takes 3–4 h. The dissociated cells can be maintained in culture for up to at least 3 weeks. Some cells expressed the neuron-specific marker microtubule-associated protein 2 and exhibited spontaneous action potentials.

1. Introduction

Neurological disorders have emerged as a predominant healthcare concern in recent years due to their severe consequences on quality of life and prevalence throughout the world. Understanding the underlying mechanisms of neurological diseases and the contribution of different types of brain cells is essential for the development of new therapeutics. Animals have been widely used in brain research as models to predict human brain response to drugs and environmental stressors. However, inherent species-specific differences in brain physiology and genetics are increasingly acknowledged [1–4]. There is a crucial need for neurological models with higher fidelity to human brains. Human-induced pluripotent stem cells (iPSCs) reprogrammed from somatic cells (e.g., fibroblasts and excreted urine cells) are able to replicate indefinitely and differentiate into virtually every cell type [5–8]. Recently developed human iPSC-derived three-dimensional (3D) cerebral organoids (Fig. 1a) from Dr. Knoblich’s lab allow more relevant in vitro systems for studying the human brain system and diseases than animal models.

Fig. 1.

Human-induced pluripotent stem cell-derived 2-month 3D cerebral organoids. (a) Morphology of cerebral organoids. (b) The image of the immunostained cerebral organoid tissue section. Ventricle-like lumens appear within the 3D organoids. The organoids display well-organized elaborate cellular laminar architectures. Neural stem cells and neurons are located in the different layers of the organoid tissues. SOX2-positive neural stem cells (green) are located on the apical side, and neurons (red) are located on the basal side (periphery) of the organoids. Blue are cell nuclei stained with Hoechst 33342

Cerebral organoids can be generated in vitro by growing iPSCs in an extracellular matrix. This culture system allows cells to differentiate into multiple brain cells (Fig. 1b) (e.g., neural stem cells, neurons, astrocytes, microglial, and endothelial cells) and develop into multiple cortical layers and multiple region-like areas (e.g., forebrain and choroid plexus) [4, 6, 9–12]. Data from our lab and others show that cerebral organoids recapitulate real brains in electrical activity, response to drugs, heterogeneous tissue structure, and functional cellular components (e.g., neurons, astrocytes, vascular cells, and microglia) with higher fidelity than widely used 2D monolayer cell culture models [4, 6, 10, 11, 13–15]. To date, cerebral organoids have been used to model several brain diseases [e.g., brain development disorders (e.g., autism), various virus infections (e.g., COVID-19 and Zika virus), and neurodegeneration (e.g., Alzheimer’s Diseases)] [4, 16, 17, 18–28]. Additionally, to address the concerns of inherent species-specific differences, many other researchers studying neurological topics have turned to the application of human iPSC-derived cerebral organoid models in modeling human brains in health and disorder and in testing drugs for their neurotoxicity and therapeutic efficacy.

Some organoid-based experiments (e.g., flow cytometry, electrophysiology property assay, and single-cell RNA sequencing) require dissociated cells. These dissociated cell-based assays can provide valuable insights into different brain cell actions in physiological and pathological conditions. Thus, the development of reliable protocols to isolate, culture, and maintain cells from cerebral organoids is highly needed and essential, particularly to studies on the cell-type-specific signaling, electrophysiological properties, and contribution of different types of cells to brain function in health and diseases. Here we present a protocol for isolation and culture of brain cells from 2-month human iPSC-derived 3D cerebral organoids. The dissociation and plating of cells from organoids takes 3–4 h. The neurons can be maintained in culture for at least 3 weeks. Some cells expressed neuron-specific marker microtubule-associated protein 2 (MAP2) and exhibited spontaneous action potentials.

2. Materials

2.1. Reagent and Medium

See Table 1.

Table 1.

Reagent and medium information

Items	Vendors	Cat.#
Hank’s Balanced Salt Solution (HBSS)	Thermo Fisher Scientific	14175095
Advanced DMEM/F12	Thermo Fisher-Scientific	12634010
Neurobasal® Medium	Thermo Fisher-Scientific	21103049
0.05% Trypsin-EDTA	Thermo Fisher Scientific	25300054
B-27® Supplement (50x)	Thermo Fisher Scientific	17504044
Fetal bovine serum (FBS)	Thermo Fisher Scientific	16000044
GlutaMAX™ Supplement	Thermo Fisher Scientific	35050061
Penicillin-Streptomycin	Thermo Fisher Scientific	15140122
DNase I, Bovine Pancreas	Sigma	260913
Corning® Matrigel® Matrix, Growth Factor Reduced	Corning	354230
Cytosine β-D-arabinofuranoside (Ara-C) (optional)	Sigma	C1768

2.2. Supplies

• 6-well plates (vWR)

• Sterile scissors (Fine Science Tools)

• 10, 200, and 1000 μl cotton-plugged pipette tips (Sorenson BioScience)

• 200 μl pipette tips without cotton plugs (vWR)

• 5, 10, and 25 ml serological pipettes (Eppendorf)

• 40 μm cell strainer (Falcon) (optional)

• 0.20 μm syringe filter (Corning)

• 15 and 50 ml centrifuge tubes (vWR)

• 1.5 ml microcentrifuge tubes (Thermo Fisher Scientific)

• Parafilm (Parafilm M)

• 70% ethanol (vWR)

• Cell culture grade water (Thermo Fisher Scientific)

• Centrifuge (Eppendorf)

• Ice

• Bead bath (Lab Armor)

• Hemocytometer (Hausser Scientific)

2.3. Preparation of Reagents, Culture Dishes/Multiple Well Plates, and Cell Culture Medium in Advance

2.3.1. DNase I Stock Solution (5 mg/ml)

Dissolve 50 mg DNase I with 10 ml HBSS, sterilize the mixed solution with 0.22 μm sterile syringe filter, aliquot the solution (500 μl/vial), and store at −20 °C.

2.3.2. Ara-C Stock Solution (5 mM)

Dissolve 10 mg Ara-C in 8.22 ml cell culture grade water, sterilize the mixed solution with 0.20 μm sterile syringe filter, aliquot the solution (1 ml/vial), and store at −20 °C.

2.3.3. Matrigel Stock Solution (2 mg/ml) (See Note 1)

1. Check the BD Quality Certificate website (http://regdocs.bd.com/regdocs/searchCOA.do) and obtain the concentration for Matrigel based on the lot and catalog numbers. Generally, the concentration of Matrigel is 7–10 mg/ml (10 ml/vial).

2. Thaw Matrigel on ice.

3. Dispense 34 ml (if Matrigel concentration is 8.8 mg/ml) of cold DMEM/F-12 using a 10 ml cold pipette into a 50 ml centrifuge tube and keep the tube on ice.

4. Dilute the Matrigel by adding 10 ml Matrigel (8.8 mg/ml) to the above centrifuge tube containing 34 ml DMEM-F12 medium to make Matrigel stock solution with the concentration of 2 mg/ml.

5. Aliquot the Matrigel stock solution (250 μl/vial) and store at −80 °C.

2.3.4. Coating Culture Dishes/Plates with Matrigel Working Solution (80 μg/ml) (See Notes 1 and 2)

1. Make Matrigel working solution (80 μg/ml): add thawed 250 μl Matrigel stock solution to 6 ml cold DMEM/F-12 and mix well. The vial may be washed with cold medium if desired.

2. Place the Matrigel^® working solution immediately to cultureware (plates or dishes). The recommended Matrigel coating volumes depending on cultureware are 1.5 ml/p60 mm dish, 60 μl/well in 96-well plate, 600 μl/well in 12-well plate, and 1 ml/well in a 6-well plate.

3. Swirl the cultureware to spread the Matrigel solution evenly until the entire surface is completely covered by the solution.

4. Incubate the coated cultureware at room temperature (15–25 °C) for at least 1 h before use. If not used immediately, the cultureware must be sealed with Parafilm to prevent evaporation of Matrigel solution and stored at 4 °C for up to 7 days after coating. Allow the stored cultureware coated with Matrigel to come to room temperature (15–25 °C) for 30 min before plating cells.

2.3.5. Cell Culture Medium (See Note 3)

1. Neuronal plating medium. Under sterile conditions, combine 445 ml of Neurobasal Medium, 50 ml FBS, and 5 ml Penicillin-Streptomycin. The plating medium is stored at 4 °C.

2. Neuronal feeding medium. Under sterile conditions, combine 490 ml of Neurobasal Medium, 10 ml B27, 5 ml Penicillin-Streptomycin, and 5 ml GlutaMAX. The feeding medium is stored at 4 °C.

3. Neuronal culture medium. Under sterile conditions, combine 50 ml neural feeding medium and 50 μl Ara-C stock solution (5 mM). The culture medium is stored at 4 °C.

3. Methods

3.1. Collect and Wash Cerebral Organoids Derived from Human iPSCs

1. To uptake the relatively large 3D organoids (Fig. 1a), 1 ml pipette tip will be modified to have a larger tip trough cutting of the first ~1 cm. Use the modified pipette tip to remove iPSC-derived 2-month organoids to a 15 or 50 ml centrifuge tube containing HBSS (2 ml/organoid) in a cell culture hood (See Note 4).

2. Allow the organoids to settle to the bottom of the tube, and then carefully remove all HBSS, without disturbing the tissues.

3.2. Tissue Dissociation

1. Add pre-warmed (37 °C) 0.05% Trypsin-EDTA (3 ml/organoid) digestion solution to the centrifuge tube containing the washed organoids using a 10 ml pipette (See Note 5).

2. Digest the organoid tissue in an incubator at 37 °C for 120 min (swirl the tube every 10 min) (See Note 6).

3. Add an equal volume of neuronal plating medium to the cell suspension to terminate the digestion (See Note 7).

3.3. Cell Trituration

1. Add DNase I stock solution to cell suspension above (10 μl/ml) to degrade extracellular DNA resulted from the lysed cells during the tissue dissociation, thereby avoiding the loss of cells from the undesired clumping (See Note 8).

2. Gently triturate organoid tissues ten times with a 10 ml serological pipette to dissociate larger aggregates (See Note 9).

3. Gently triturate dissociated organoids another ten times with a 10 ml serological pipette tipped with a 200 μl tip without cotton plug to dissociate small aggregates.

4. (Optional for cell culture) Filter the triturated cell suspension through 40 μm cell strainer; that way, incompletely dissociated cell clumps will be removed (See Note 10).

5. Centrifuge triturated cell suspension at 80 × g for 5 min at room temperature.

6. Discard the supernatant after the centrifugation and suspend the cell pellet with 5 ml neuronal plating medium.

3.4. Cell Plating and Culturing

1. Count the number of dissociated cells using a hemocytometer. This protocol maintains a high yield of live cells, estimated to be around 4 × 10⁶ cells per organoid.

2. Plate cells in the Matrigel-coated cultureware and culture cells with neuronal plating medium (2.6 ml/well in 6-well plate). Optimal cell plating density must be determined for the specific cell application.

3. Three hours after plating, replace the neuronal plating medium with neuronal feeding medium (See Note 11).

4. (Optional) After 24 h, replace half of the old media with the same volume of neuronal culture medium (neuronal feeding medium containing 5 μM Ara-C) for inhibition of cell proliferation.

5. Neuronal processes can be visible on day 1, and an extensive neural network is formed in the culture on day 6. Some cells express neuron-specific marker MAP2 (Fig. 2) and exhibit spontaneous action potential (Fig. 3).

6. Change the medium twice a week (See Note 12).

Fig. 2.

The dissociated cells from 2-month cerebral organoids. (a) The organoid-derived cells on a hemocytometer immediately after dissociation. (b) A phase contrast image of cells in the culture 6 days after the dissociation from organoids. Many cells exhibit neuron-like morphology with a small round cell body extending long projections. (c) An immunofluorescence image displays that some dissociated cells expressed neuron-specific marker microtubule-associated protein-2 (MAP2, red). Blue are cell nuclei

Fig. 3.

Two-month cerebral organoids possess action potentials. (a) Cells dissociated from cerebral organoids were plated on wells containing electrodes (scale bar = 400 μm). (b) The dissociated neurons from organoids displayed spontaneous action potential firing through a multiple electrode array assay. Representative action potential amplitude is shown on the left, and spontaneous spike frequency is shown on the right

4. Notes

1. Warm up 0.05% Trypsin-EDTA digestion solution in a 37 °C bead bath or incubator for 30 min prior to use.

2. Gently handling organoid tissues and dissociated cells throughout the experiment is critical to yield dissociated cells with high quality.

3. Aliquot culture medium and store at −20 °C if the medium is not used within long period. After thawing the aliquots, use within 2 weeks. Do not re-freeze.

4. Make sure to always keep Matrigel on ice when thawing and handling to prevent it from gelling.

5. Successful generation and maintenance of dissociated cells requires the use of a suitable matrix to allow attachment of cells. We generally use Matrigel for coating cultureware. Poly-L-ornithine/laminin-coated cultureware can be also used for neuron culture as previously described https://www.neuvitro.com/poly-l-ornithine-coating.

6. Recommended dissociation time for 2-month organoids is 120 min, but optimal dissociation conditions must be determined based on the organoids with different age, size, and application.

7. Recommended dilution factor for DNase I stock solution is 1:100, but optimal DNase I working concentration must be determined for organoids with different sizes.

8. Avoid bubbles during trituration. Limit the trituration within 20 strokes.

9. This step is necessary for obtaining single-cell suspension if the dissociated cells are used for flow cytometry or single-cell RNA sequencing assays.

10. Alternative medium and protocols can be used for termination of organoid digestion, including the following: (a) directly add FBS to the cell suspension including 0.05 trypsin/EDTA solution and (b) add an equal volume of other medium (e.g., DMEM and DMEM/F12) containing 10% FBS to the cell suspension.

11. When changing medium, use transfer pipettes, but avoid using the vacuum system.

12. Use the cultured cells for the study within 21 days after plating. Long culture might result in cell aggregation or cell detachment from the cultureware.