A Technique to Generate a 2D Monolayer of Cerebellar Cells from Induced Pluripotent Stem Cells

Protocolo

1. Experimental preparation

1. Prepare basement membrane matrix (BMM)-coated plates.
   NOTE: BMM solidifies more quickly at warmer temperatures. Plates must be prepared rapidly and immediately placed at 4 °C for storage.
   1. Thaw the BMM (see Table of Materials) on ice, at 4 °C for at least 2 h, or overnight.
   2. Mix Dulbecco's Modified Eagle Medium (DMEM)/F12 and BMM to a final concentration of 80 µg/mL. Distribute the BMM solution in tissue culture dishes (1 mL for 35 mm and 2 mL for 60 mm dishes) and incubate at 37 °C for at least 1 h or overnight before plating the cells.
      ​NOTE: The unused dishes can be stored at 4 °C for 2 weeks.
2. Prepare poly-L-ornithine/laminin (PLO/laminin)-coated plates.
   1. Prepare 20 µg/mL PLO (see Table of Materials) in sterile Dulbecco's Phosphate Buffered Saline (DPBS+/+) and add 1 mL to each well of a 6-well plate. Incubate overnight at 37 °C in the incubator.
   2. The following day, aspirate the PLO with a vacuum aspirator and wash two times with DPBS+/+. Air dry in the hood.
      ​NOTE: Air-dried plates can be stored at 4 °C for up to 2 weeks, wrapped in aluminum foil, for future use.
   3. Prepare 10 µg/mL laminin (see Table of Materials) in DPBS+/+ and add 1 mL to each well of a 6-well plate. Incubate at least for 3 h or overnight at 37 °C in the incubator.
   4. Aspirate the laminin with a vacuum aspirator and add either 1 mL of medium or sterile DPBS+/+.
      ​NOTE: The laminin coating must not dry out. PBS or an appropriate culture medium should be added immediately to prevent this. Coated plates can be stored at 4 °C for up to 2 weeks.
3. Prepare PSC passaging solution.
   1. Dissolve 11.49 g of potassium chloride (KCl) and 0.147 g of sodium citrate dihydrate (HOC(COONa) (CH₂COONa)₂*2H₂O) in 400 mL of sterile cell culture grade water (see Table of Materials).
   2. Measure the volume of the solution and record it as the initial volume (Vi).
   3. Measure the osmolarity and adjust it to 570 mOsm by adding sterile cell culture grade water using the formula Vi × Oi = Vf × 570 mOsm.
   4. Filter-sterilize the solution with a 0.20 µm filter and make 10 mL aliquots. Store the aliquots at room temperature (RT) for up to 6 months.
4. Prepare pluripotent stem cell (PSC) medium.
   ​NOTE: iPSCs are maintained in a medium containing heat-stable Fibroblast growth factor 2 (FGF2) (see Table of Materials), providing a weekend-free feeding schedule. Other commercially available PSC media have not been tried for this protocol.
   1. Thaw the PSC medium supplement overnight at 4 °C.
   2. Add 10 mL of PSC medium supplement to 500 mL of basal PSC medium. Make 25 mL aliquots and store at −20 °C.
      ​NOTE: Frozen aliquots can be stored at −20 °C for 6 months. Thawed aliquots must be stored at 4 °C and used within 2 weeks. Cells can be frozen for long-term storage in a PSC medium with 10% (v/v) dimethyl sulfoxide (DMSO).
5. Prepare PSC thawing medium.
   1. Supplement PSC medium with 50 nM chroman, 1.5 µM emricasan, 1x polyamine supplement, and 0.7 µM trans-ISRIB (CEPT cocktail) (see Table of Materials).
   2. Filter-sterilize with a 0.20 µm filter and store at 4 °C for up to 4 weeks.
6. Prepare pulled glass pipettes.
   1. Pull 22.9 cm (9 in) glass Pasteur pipettes into two pieces above a Bunsen burner, ~2 cm below the neck, creating two pipettes, with the thinner side being ~4 cm shorter than the other.
   2. With the help of the flame, bend the tips of the pulled side to create a smooth "r".
   3. Place the pulled pipettes in autoclave sleeves and autoclave for sterilization.
7. Prepare cerebellar differentiation medium (CDM).
   1. Mix Iscove's Modified Dulbecco's Medium (IMDM) and Ham's F12 nutrient mix in a 1:1 ratio. Supplement the mix with 1x L-alanine-L-glutamine supplement, 1% (v/v) chemically defined lipid concentrate, 0.45 mM 1-thio-glycerol, 15 µL/ mL apo-transferrin, 5 mg/mL bovine serum albumin (BSA), and 7 µg/mL insulin (see Table of Materials).
   2. Filter-sterilize with a 0.20 µm filter, store at 4 °C, and use within 1 month.
8. Prepare cerebellar maturation medium (CMM).
   1. Supplement neurobasal medium with 1x L-alanine-L-glutamine supplement and 1x N-2 supplement (see Table of Materials).
   2. Filter-sterilize with a 0.20 µm filter, store at 4 °C, and use within 2 weeks.

2. Feeder-free iPSC culture

1. Thaw the cells following the steps below.
   1. Place a BMM plate (step 1.1) into the 37 °C incubator to gel for at least 1 h or overnight before thawing the cells.
   2. Pre-warm 10 mL of PSC thawing medium (step 1.5) at 37 °C.
   3. Transfer the cryovial with cells from liquid nitrogen to a water bath at 37 °C.
      ​NOTE: To avoid generating a source of contamination in the cell culture space, using a standard water bath is not recommended. Instead, fresh water can be heated in a small beaker to 37 °C to generate a one-time-use water bath.
   4. When there is a small piece of ice left in the tube, remove it from the water bath, dry the tube, and spray with 70% ethanol. Transfer the tube to the hood and use a 2 mL or 5 mL serological pipette (see Table of Materials) to gently transfer the cells to a 15 mL conical tube.
   5. Add 8 mL of PSC thawing medium to the cells dropwise while swirling the conical tube. Centrifuge at 200 x g for 5 min at RT.
   6. Carefully aspirate the supernatant with a vacuum aspirator without disturbing the cell pellet. Resuspend the cells in 2 mL of PSC thawing medium.
   7. Aspirate the BMM from the plate and add the resuspended cells dropwise all over the plate for even distribution.
   8. Place the plate in the incubator at 37 °C, 5% CO₂. Refresh the medium the next day with PSC medium. After that, change the medium daily.
2. Maintain the cells following the steps below.
   1. Pre-warm the necessary volume of PSC medium at 37 °C (e.g., 1 mL of medium per 35 mm plate).
   2. Investigate the plates for differentiated cells or colonies and remove the differentiated cells with a pulled glass pipette (step 1.6).
      NOTE: iPSC colonies have smooth edges with morphologically identical cells.
   3. Change the spent medium daily and passage every 3-4 days or when 70% confluency is reached.
3. Passage the cells.
   1. Place the necessary amount of BMM plates into the incubator for at least 1 h or overnight before passaging. Pre-warm the necessary amount of PSC medium (step 1.3) at 37 °C.
   2. Using a pulled glass pipette, clean off any differentiated cells or colonies.
   3. Aspirate the spent medium with a vacuum aspirator and add PSC dissociation medium, 1 mL per 35 mm dish. Incubate at 37 °C for 1-3 min.
      ​NOTE: If colonies are lifting off in the PSC passaging solution before adding PSC medium, the incubation time can be reduced.
   4. Aspirate the PSC passaging solution and add pre-warmed PSC medium.
   5. Using a sterile 200 µL pipette tip, scratch lines parallel to each other in one direction, turn the plate 90°, and make a second set of scratch lines perpendicular to the previous ones (making a cross-hatched pattern across the plate).
   6. Aspirate the BMM solution from the set plates. Collect the colonies using a serological pipette and distribute them to new BMM plates.
   7. Incubate at 37 °C, 5% CO₂. Change the medium daily.
4. Freeze the cells.
   1. Prepare cryovials by labeling the content and sterilize under ultraviolet (UV) light in the hood (see Table of Materials) for 30 min.
   2. Prepare PSC freezing medium by supplementing PSC medium with 10% (v/v) DMSO. Follow steps 2.3.2-2.3.5.
   3. Transfer the cells to a 15 mL conical tube with a 5 mL serological pipette and centrifuge at 200 x g for 5 min at RT.
   4. Carefully aspirate the medium and resuspend the cell pellet in PSC freezing medium.
   5. Distribute into cryovials. Place the vials into a freezing container and place the container in a −80 °C freezer.
   6. The following day, transfer the cryovials to liquid nitrogen for long-term storage.

3. Cerebellar differentiation

NOTE: Before starting the differentiation, iPSCs are passaged to six 35 mm dishes and are ready for the differentiation when they are at 70% confluency. Each 35 mm plate will be transferred to one well of the 6-well plate.

1. On day 0, lift the healthy iPSC colonies for EB formation.
   1. Add 1 mL of CDM (step 1.7) supplemented with 10 µM Y-27632 and 10 µM SB431542 (see Table of Materials) to each well of a 6-well ultra-low attachment (ULA) plate and place in the incubator until the lifted colonies are ready to be added to the wells.
   2. Clean the differentiated cells using a pulled glass pipette. Aspirate the medium and add 1 mL of PSC passaging solution for each 35 mm dish. Incubate for 3 min at 37 °C, aspirate, and add 2 mL of CDM supplemented with 10 µM Y-27632 and 10 µM SB431542.
      NOTE: If colonies are lifting off in the PSC passaging solution before adding CDM, the incubation time can be reduced.
   3. Under a transmitted-light inverted microscope, gently lift the colonies using the bent edge of the pulled glass pipette using 4x magnification. Once every colony is lifted, gently transfer all to one well of the 6-well ULA plate using a 10 mL serological pipette. Repeat this process for each iPSC plate. Incubate the cells at 37 °C with 5% CO₂.
      NOTE: If the colonies are too large or are merged, they can be sliced with the tip of the pulled glass pipette to create smaller EBs.
2. On day 2, add FGF2 to each well to a final concentration of 50 ng/mL.
   NOTE: The FGF2 used for cerebellar differentiation is not heat-stable. This protocol has not been tested with heat-stable FGF2.
3. On day 7, do a 1/3 medium change. For 3 mL of total medium in a well, with a 1,000 µL pipettor, gently aspirate 1 mL of spent medium and replace it with 1 mL of fresh CDM. Incubate the EBs for 7 days.
4. On day 14, gently aspirate nearly all the spent medium using a 1,000 µL pipettor. To ensure minimal damage to the EBs, swirl the plate to gather all the EBs in the center of the plate, and then tilt the plate and aspirate slowly from the edge.
   1. As the medium amount decreases, slowly lay the plate flat and continue to aspirate. Leave enough medium to avoid drying the EBs out. Afterward, add 3 mL of fresh CDM supplemented with 10 µM Y-27632. Transfer the EBs using a 10 mL serological pipette to a PLO/laminin-coated dish (step 1.2).
      NOTE: Depending on the downstream application, the EBs can be transferred to a 6-well PLO/laminin plate or single EBs can be transferred to a single well of a PLO/laminin-coated 24-well plate or coverslip.
5. On day 15, aspirate the medium and replace it with fresh CDM.
   NOTE: It is important to add enough medium to the wells to ensure enough medium between feedings as, over time, there will be evaporation (e.g., for a well in a 6-well plate, add 3 mL of medium). If the medium has started to acidify (turn clear and yellow), refresh the medium even if it is not on the feeding schedule until the end of the protocol.
6. On day 21, aspirate the spent medium and replace it with CMM. On day 28, change the medium with fresh CMM. On day 35, harvest the cells for further applications.

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Materiais

Name	Company	Catalog Number	Comments
10 mL Serological pipette	Fisher Scientific	13-678-26D
1-thio-glycerol	Sigma	M6145
2 mL Serological pipette	Fisher Scientific	13-678-26B
250 mL Filter Unit, 0.2 µm aPES, 50 mm Dia	Fisher Scientific	FB12566502
35 mm Easy Grip Tissue Cluture Dish	Falcon	353001
5 mL Serological pipette	Fisher Scientific	13-678-25D
60 mm Easy Grip Tissue Culture Dish	Falcon	353004
6-well ultra-low attachment plates	Corning	3471
9" Disposable Pasteur Pipets	Fisher Scientific	13-678-20D
Apo-transferrin	Sigma	T1147
Bovine serum albumin (BSA)	Sigma	A9418
Cell culture grade water	Cytiva	SH30529.02
Chemically defined lipid concentrate	Gibco	11905031
Chroman 1	Cayman	34681
Class II, Type A2, Biological safety Cabinet	NuAire, Inc.	NU-540-600	Hood, UV light
Costar 24-well plate, TC treated	Corning	3526
Costar 6-well plate, TC treated	Corning	3516
DMEM	Gibco	11965092
DMEM/F12	Gibco	11320033
DMSO (Dimethly sulfoxide)	Sigma	D2438
DPBS+/+	Gibco	14040133
Emricasan	Cayman	22204
Epi5 episomal iPSC reprogramming kit	Life Technologies	A15960
Essential 8-Flex	Gibco	A2858501	PSC medium with heat-stable FGF2
Fetal bovine serum - Premium Select	Atlanta Biologicals	S11150
FGF2	Peprotech	100-18B
GlutaMAX supplement	Gibco	35050061	L-alanine-L-glutamine supplement
Ham's F12 Nutrient Mix	Gibco	11765054
HERAcell VIOS 160i CO2 incubator	Thermo Scientific	50144906
IMDM	Gibco	12440053
Insulin	Gibco	12585
Laminin Mouse Protein	Gibco	23017015
Matrigel Matrix	Corning	354234	Basement membrane matrix
MEM-NEAA	Gibco	11140050
Mini Centrifuge	Labnet International	C1310	Benchtop mini centrifuge
N-2 supplement	Gibco	17502-048
Neurobasal medium	Gibco	21103049
PBS, pH 7.4	Gibco	10010023
PFA 16%	Electron Microscopy Sciences	15710
Polyamine supplement	Sigma	P8483
Poly-L-Ornithine (PLO)	Sigma	3655
Potassium chloride	Sigma	746436
SB431542	Sigma	54317
See through self-sealable pouches	Steriking	SS-T2 (90x250)	Autoclave pouches
Sodium citrate dihydrate	Fisher Scientific	S279-500
Syringe filters, sterile, PES 0.22 µm, 30 mm Dia	Research Products International	256131
Trans-ISRIB	Cayman	16258
Vapor pressure osmometer	Wescor, Inc.	Model 5520	Osmometer
Y-27632	Biogems	1293823