Introduction
This article describes how one of our customers who applied MeltonVEC-Sendai iPSC Reprogramming Kit on the transduction of newborn fibroblasts into iPSCs successfully. There are detailed materials and methodology, highly illustrated results in this informative article.
Details
(Work flow of fibroblast reprogramming into iPSCs)
Step 1: Preparation of Reagents
1. Culture Medium: Fibroblast Growth Medium (MD-0011 Fibroblast Growth Medium)
iPSC Culture Medium (mTeSR Plus Medium, STEMCELL, 100-0276)
2. Matrix Gel: Matrigel (Corning 354230)
3. iPSC Reprogramming kit-MeltonVEC™-Sendai iPSC Reprogramming Kit (Normal)
4. DPBS (Dulbecco's phoshphate buffered saline)
5. 0.05% Trypsin/EDTA (0.25% Trypsin/EDTA diluted 5 times with DPBS)
6. mTeSR Plus Basal Medium (Stemcell Technologies, Canada)
DAY -1 （2024.09.10）
Day –1: Preparing Cells for Transduction
On the day before transduction, plate fibroblasts (from newborn babies) at three different densities (1×10^5, 2×10^5, 3×10^5 cells/well) into three wells of a 6-well plate. (One of the wells with the same density will be used on the day of transfection to count the cells in order to calculate the volume of virus to be added).
Note: Each MeltonVEC™-Sendai iPSC Reprogramming Kit provides enough virus to transduce cells in at least five wells of a 6-well plate. We recommend using the entire volume of virus in each tube of the kit
Note: We suggest achieving approximately 30-60% confluence on the day of transduction. Over-confluence can lead to reduced transduction efficiency. If your cells become overly confluent during the culture process, we recommend replating them to reach 30-60% confluence.
DAY 0 - Transduction (2024.09.11)
Calculation of Cell Density:
Use one well from each of the three plated densities for live cell counting. (The cells in this well will not be transfected and are used to estimate the cell numbers in the other plated wells).
Digestion and Counting Steps:
A. Digest the cells in the 6-well plate using 0.05% Trypsin/EDTA and incubate at room temperature. Once the cells begin to detach (after 1-3 minutes), add 1 mL of fibroblast growth medium to each well to stop the digestion, and collect the cells in a 15 mL conical centrifuge tube.
B. Perform trypan blue staining to count the live cells and calculate the volume of virus to be added based on the cell count.
Calculation of MOI (Multiplicity of Infection):
MeltonVEC™-Sendai iPSC Reprogramming Kit（KOS MOI=10，hc-Myc MOI=10，hKlf4 MOI=3）
CIU (Cell Infecting Unit)
Virus Reagent Thawing and Cell Transduction with Virus:
A. Take out MeltonVEC iPSC Reprogramming Kit from -80°C fridge. Thaw one vial at a time by immersing the bottom of the vial in a 37°C water bath for 5-10 seconds, then remove the vial from the water bath and allow it to thaw at room temperature. After thawing, briefly centrifuge the vial and place it on ice immediately.
B. Add the calculated volume from the three vials of the MeltonVEC iPSC Reprogramming Kit to 1 mL of fibroblast growth medium pre-warmed to 37°C. Prepare two portions for each of the three different cell densities, and add 6 µg/mL of polybrene to one of the portions. Gently pipette up and down to ensure the solution is thoroughly mixed. Proceed to the next step within 5 minutes.
C. Remove the culture medium from the fibroblasts in the 6-well plate, and add the 1 mL of reprogramming virus mixture prepared in step B to each well containing cells.
D. Centrifuge the entire plate at 1000 rpm for 30 minutes, then gently return it to the 37°C incubator with 5% CO2 and incubate overnight.
DAY 1- DAY 6 （2024.09.12 - 20240.09.17）
Changing the Medium and Culturing the Cells:
A. 24 hours after transduction, replace the medium with fresh fibroblast growth medium.
Note: Depending on the cell types, one may observe some cytotoxicity 24-48 hours post-transduction, which could affect more than 50% of the cells. This indicates a high uptake of the virus. We recommend continuing to culture the cells.
B. Continue culturing the cells for an additional 6 days, changing the medium with fresh fibroblast growth medium every other day.
Note: Depending on the cell types, you may observe high cell density before day 5. We do not recommend passaging your cells before day 7 post-transduction. If the culture becomes very dense, replace the medium with fresh fibroblast growth medium daily. Do not passage the cells after transfection. If cell density is too high, change the medium daily.
DAY 1（2024.09.12）
DAY 7 - Pre-Digestion (2024.09.18)
Day 7: Culture the transducted cells on a Matrigel-coated dish.
1. Coat seven wells of a 6-well plate with Matrigel.
2. Seven days post-transduction, digest the fibroblasts with 100,000 cells (without polybrene), 200,000 cells (without polybrene), and 300,000 cells (without polybrene). Then, reseed them at densities of 1×10^5 and 2×10^5 cells per well into the Matrigel-coated wells. For the 100,000 cells with polybrene, reseed at a density of 2×10^5 cells per well.
a) Remove the medium from the fibroblasts and wash the cells once with DPBS.
b) Digest the cells with 0.05% trypsin/EDTA and incubate at room temperature for 1–3 minutes.Add 2 ml of fibroblast medium to neutralize the trypsin, then collect the cells into a 15 mL centrifuge tube.
Note: Since the cells are very sensitive to trypsin at this stage, minimize the exposure time to trypsin and incubate the cells at room temperature.
3. Centrifuge the cells at 200×g for 4 minutes, aspirate the medium, and gently resuspend the cells in an appropriate amount of fibroblast medium. Be gentle during this process.
4. Use trypan blue to count the viable cells, and add 2×10^4 to 1×10^5 cells per well in the Matrigel-coated 6-well plate. Reseed the wells at densities of 1×10^5 and 2×10^5 cells per well in the Matrigel-coated 6-well plate. Set aside some of the cell pellet for freezing at -80°C.
Note: When using feeder-free conditions, the reprogramming efficiency is usually lower, so the number of cells added per well should be correspondingly increased. We recommend using at least two to four different densities per well. Depending on the cell types, place the majority of the cells on the same plate to ensure there are enough colonies.
Note: It is highly recommended to reserve some cells for RNA extraction at this point, to serve as a positive control for RT-PCR or qPCR detection of the MeltonVEC vector. Including this positive control is crucial when performing MeltonVEC detection.
DAY 8- DAY28 （2024.09.19- 2024.10.09）
Medium Change and Cell Observation
1. Start changing the medium to mTeSR Plus on Day 8, and thereafter, replace with fresh mTeSR Plus medium daily.
2. From Day 8 onwards, observe the plates under a microscope every other day to check for the appearance of iPSC clusters, indicating that the cells have been reprogrammed.
Note: For neonatal fibroblasts, colonies typically start forming around Day 12 post-transduction. Depending on the cell types, it may take up to 4 weeks of culture to observe iPSC clusters.
3. After three to four weeks post-transfection, the colonies should grow to a suitable size for transfer.
Note: harvest the colonies before three weeks to avoid differentiation.
4. Manually pick undifferentiated iPSC colonies (see next page) and transfer them to a prepared Matrigel-coated 6- or 12-well plate for further expansion or analysis.
DAY 8
DAY 9
DAY 10
DAY 14 (2024.9.25)
DAY 15 (2024.9.26)
Alkaline Phosphatase Staining（2024.9.30)
