Dec 16, 2024
Fibroblasts into iPSCs reprogramming protocol using Sendai virus
- yuchae lee1
- 1KAIST
Protocol Citation: yuchae lee 2024. Fibroblasts into iPSCs reprogramming protocol using Sendai virus. protocols.io https://dx.doi.org/10.17504/protocols.io.ewov1ddm2vr2/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: December 15, 2024
Last Modified: December 16, 2024
Protocol Integer ID: 115569
Abstract
This protocol is based on feeder free on vitronectin-coated culture dish.
Preparation
Preparation
Fibroblast culture medium : @4°C lasts for 1 month. A total of 500 mL:
- 435 ml DMEM (High glucose, GlutaMAX)
- 50 ml Fetal bovine serum (FBS)
- 5 ml Penicillin-Streptomycin (Optional)
- 5 ml MEM NEAA (100X)
- 5 ml Sodium pyruvate
- 0.5 ml beta-mercaptoethanol
- Sterilized by filtration through a 0.22 µm filter
Pre-warm to room temperature (20 °C-25 °C) before use.
Store Fibroblast Culture Medium at 2 °C-8 °C for up to 4 weeks.
(optional) ReproTeSR‱ medium for maintenance of adherent cells undergoing reprogramming:
Preparing 500 mL of mTeSR. If preparing other volumes, adjust accordingly.
Combine the following:
- 100 mL 5x mTeSR supplement (stored ≥-20°C and thawed overnight in TC fridge).
Pre-warm to room temperature (23 °C – 25 °C, not 37 °C) before use.
Reprogramming Process
Reprogramming Process
Note
The major steps required for reprogramming of the primary human dermal fibroblasts using the ‱ CytoTune‱-iPS 2.0 Sendai Reprogramming Kit to generate iPSCs cultured feeder-free on rhLaminin521-coated culture dishes are shown below. Note that the timeline is provided as a guideline for experimental planning; the actual timeline can vary based on the cell type and experimental conditions (e.g. dynamics of colony appearance):
Day -2 : Prepare the cells for transduction
Plate fibroblasts on Matrigel coated 6-well plate at the appropriate density to achieve between 2 × 105 –3 × 105 cells per well on the day of transduction (Day 0).One of the wells will be used to count cells for viral volume calculations.
Culture the cells for two more days, ensuring the cells have fully adhered and extended.
Day 0 : Perform transduction
On the day of transduction, warm 1 mL of fibroblast medium in a water bath for each well to be transduced.
Harvest the cells from one wellto perform a cell count. These cells will not be transduced, but will be used to estimate the cell number in the other well(s) plated in Step 5.
Remove the cells from the 6-well plate using 0.5 mL of 0.05% trypsin/EDTA following the procedure recommended by
the manufacturer and incubatingat room temperature. When the cells have rounded up (1–3 minutes later), add 1 mL of fibroblast medium into each well, and collect the cells in a 15-mL conical centrifuge tube.
Count the cells using the desired method (e.g., Countess‱Automated Cell Counter), and calculate the volume of each virus needed to reach the target MOI using the live cell count and the titer information on the CoA.
Remove one set of CytoTune‱ 2.0 Sendai tubes from the –80°C storage. Thaw each tube one at a time by first immersing the bottom of the tube in a 37°C water bath for 5–10 seconds, and then removing the tube from the water bath and allowing it to thaw at room temperature. Once thawed,briefly centrifuge the tube and place it immediately on ice.
Add the calculated volumes of each of the three CytoTune‱ 2.0 Sendai tubes to 1 mL of fibroblast medium, pre-warmed to 37°C. Ensure that the solution is thoroughly mixed by pipetting the mixturegently up and down. Complete the next step within 5 minutes.
Aspirate the fibroblast medium from the cells, and add the reprogramming virus mixtureprepared in Step 8 to the well containing the cells. Incubate the cells overnight in a 37°C incubator with a humidified atmosphere of 5% CO2.
Day 1 : Replace the medium with fresh complete fibroblast medium to remove the CytoTune‱ 2.0 Sendai reprogramming vectors. Culture the cells for 6 more days, changing the spent medium with fresh
fibroblast medium every other day
Day 7 : Plate transduced cells on vitronectin-coated culture dishes
Coat a sufficient number of tissue culture dishes (e.g. 6-well, 60-mm, or 100-mm) with vitronectin or Geltrex.
Seven days after transduction, fibroblast cells are ready to be
harvested and plated on vitronectin-coated culture dishes. Remove the medium from the fibroblasts, and wash cells once with D-PBS.
To remove the cells from the 6-well plate, use 0.5 mL of 0.05% trypsin/EDTA following the procedure recommended by
the manufacturer and incubate at room temperature. When the cells have rounded up (1–3 minutes later), add 2 mL of fibroblast medium into each well, and collect the cells in a 15-mL conical centrifuge tube
Centrifuge the cells at 200 × g for 4 minutes, aspirate the medium, and re-suspend the cells in an appropriate amount of fibroblast medium.
Count the cells using the desired method (e.g., Countess‱Automated Cell Counter), and seed the vitronectin-coated culture dishes with 2 × 104 –1 × 105 cells per well of a 6-well and incubate overnight in a 37°C incubator with a humidified atmosphere of 5% CO2.
Day 8-28: Feed and monitor the cells
24 hours later, change the medium to complete Essential 8‱Medium), and replace the spent medium every daythereafter.
Starting on Day 8, observe the plates every other day under a microscope for the emergence of cell clumps indicative of reprogrammed cells.
Three to four weeks after transduction, colonies should have grown to an appropriate size for transfer. When the colonies are ready for transfer, perform live staining using Tra1-60 or Tra1-81 for selecting reprogrammed colonies.
Manually pick undifferentiated iPSC colonies and transfer them onto prepared vitronectin-coated 6-or 12-well culture plates for further expansion or analysis.
Pick iPSC colonies
Pick iPSC colonies
Place the culture dish containing the reprogrammed cells under an inverted microscope and examine the colonies under 10X magnification. Mark the colony to be picked on the bottom of the culture dish.
Transfer the culture dish to a sterile cell culture hood (i.e., biosafety cabinet) equipped with a stereomicroscope.
Using a 25 gauge 1½ inch needle, cut the colony to be picked into 5–6 pieces in a grid-like pattern.
Using a 200 μL pipette, transfer the cut pieces onto a vitronectin-coated 12-or 6-well culture plate containing complete Essential 8‱ Medium.
Incubate the vitronectin-culture plate containing the picked colonies in a 37°C incubator with a humidified atmosphere of 5% CO2.
Allow the colonies to attach to the culture plate for 48 hours before replacing the spent medium with fresh complete Essential 8‱ Medium. After that, change the medium every day.
When the colonies cover ~85% of the surface area of the culture vessel, they are ready for passaging. Passage the colonies using 0.5 mM EDTA prepared in Dulbecco's Phosphate-Buffered Saline (DPBS) without calcium or magnesium.
Continue to culture, expand, and maintain the reporgrammed colonies in complete Essential 8‱ Medium until you have frozen cells from two 60-mm plates.