2 Material and methods
2.2 Methods
2.2.4 Cell culture methods
2.2.4.10 Generation of mouse ES cell lines
2.2.4.11.2 Procedure of reprogramming
The procedure of the “Feeder Free” mRNA/miRNA Reprogramming is illustrated in Tab. 1.
The reprogramming process was performed at 37°C, 5% CO2 and 5% O2. All media were equilibrated under these conditions for at least 2 h before their usage.
Tab. 1: Overview oft the procedure of “Feeder Free” mRNA/miRNA Reprogramming.
day action medium
d-3 preparation of matrigel coated
plates
-d-2 seeding of target cells appropriate cell culture medium
d-1 miRNA transfection NuFF-conditioned Pluriton™ Medium supplemented with bFGF to finally 20 ng/ml
d0 4 h preincubation NuFF-conditioned Pluriton™ Reprogramming Medium
mRNA transfection
-d1
3 h preincubation NuFF-conditioned Pluriton™ Reprogramming Medium
mRNA transfection
-medium change after 4 h NuFF-conditioned Pluriton™ Reprogramming Medium
d2 / d3 mRNA transfection
-medium change after 4 h NuFF-conditioned Pluriton™ Reprogramming Medium
d4 mRNA/miRNA co-transfection
-medium change after 4 h NuFF-conditioned Pluriton™ Reprogramming Medium
d5 + mRNA transfection
-medium change after 4 h NuFF-conditioned Pluriton™ Reprogramming Medium d: day of reprogramming; h: hours; d5 +: d5 of reprogramming until the emergence of hiPSC colonies.
2.2.4.11.2.1 Seeding of target cells
At day -2 of reprogramming target cells were seeded on matrigel coated 6-well vessels (2.2.4.1.2), which were freshly prepared the day before. For reprogramming of slowly proliferating cell lines such as the used scrotal human fibroblasts (SHFs; 2.2.4.3.8) were seeded in a high cell density of 100.000 cells per 6-well vessel, whereas fast proliferating cell lines were seeded in a cell density of 20.000 cells per 6-well vessel. Target cells were seeded in appropriate culture medium (2.1.11.1) and attached overnight at 37°C, 5% CO2 and 5% O2.
2.2.4.11.2.2 miRNA transfection
At day -1 of reprogramming target cells were transfected with miRNA cluster 302-367 (2.2.4.11.1.5). Cell were washed once with PBS and covered with 2 ml per 6-well vessel equilibrated NuFF-conditioned Pluriton™ Medium (2.2.4.11.1.3) supplemented with bFGF (= 4 µl P™S in 10 ml medium) to a final concentration of 20 ng/ml.
The following components were needed for the miRNA transfection of cells per one 6-well vessel:
All components of each tube were pipetted together and mixed carefully. The content of tube 2 was transferred in tube 1, mixed carefully by pipetting and further incubated for 15 min at room temperature. The 100 µl transfection mixture was mixed carefully by pipetting the solution three times up and down and subsequently distributed on the target cells of one 6-well vessel in a dropwise fashion. The 6-well plate was gently rocked from side to side and front and back to distribute the transfection mix across the well. Cells were further cultured at 37°C, 5% CO2 and 5% O2.
2.2.4.11.2.3 mRNA transfection series
In order to maintain a constant level of delivered mRNA in the cells, the mRNA transfection had to be performed always at the same time.
At day 0 of reprogramming target cells were transfected with mRNA (2.2.4.11.1.6). Before mRNA transfection the medium was discarded without washing with PBS and directly replaced with NuFF-conditioned Pluriton™ Reprogramming Medium (2.2.4.11.1.4). The cells were incubated for 4 h at 37°C, 5% CO2 and 5% O2 before mRNA transfection. The following components were needed for the mRNA transfection of cells per one 6-well vessel:
The transfection mix was prepared like for miRNA transfection (2.2.4.11.2.2) and distributed on the preincubated target cells in a dropwise fashion. The 6-well plate was gently rocked from side to side and front and back to distribute the transfection mix across the well. Cells were further cultured at 37°C, 5% CO2 and 5% O2.
The next day (day 1 of reprogramming) 3 h before mRNA transfection the medium was discarded without washing with PBS and directly replaced with NuFF-conditioned Pluriton™
Reprogramming Medium. The cells were incubated for 3 h at 37°C, 5% CO2 and 5% O2 before mRNA transfection, which was performed as described above. Medium was replaced with NuFF-conditioned Pluriton™ Reprogramming Medium 4 h after mRNA transfection and cells were further cultured at 37°C, 5% CO2 and 5% O2.
On day 2 and day 3 the preincubation was omitted and the mRNA transfections as well as medium change 4 h after transfection were performed as on day 1 of reprogramming.
A mRNA/miRNA co-transfection was performed on day 4 of reprogramming. The following components were needed for the co-transfection of cells per one 6-well vessel:
The transfection mix was prepared as already described and distributed on the cells in a dropwise fashion. The 6-well plate was gently rocked from side to side and front and back to distribute the transfection mix across the well. Cells were further cultured at 37°C, 5% CO2 and 5% O2. A medium change with NuFF-conditioned Pluriton™ Reprogramming Medium was performed 4 h after co-transfection and cells were further cultured at 37°C, 5% CO2 and 5% O2.
On the following days, cells were further transfected with mRNAs as described above until human iPSC colonies emerged.
2.2.4.11.2.4 Splitting of target cells during the reprogramming process
If target cells exhibited a high proliferation rate during reprogramming, they needed to be split to prevent an over-confluence of cells, which would reduce the reprogramming efficiency because of a decreased mRNA delivery. If splitting of target cells was necessary, it was preferentially performed between day 6 and 8 of the transfection series and cells were replated in a lower cell density.Cells, which had to be split, were transfected with mRNA and the medium was changed 4 h after transfection as already described (2.2.4.11.2.3). After 2 h at 37°C, 5% CO2 and 5% O2, cells were washed once with PBS and covered with 0.5 ml Trypsin/EDTA. After 10 min at 37°C, 5% CO2 and 5% O2, cell detachment and dissociation were stopped by the addition of 0.5 ml FB medium. Detached cells were transferred with a serological pipette in a 15 ml falcon tube containing 2 ml NuFF-conditioned Pluriton™ Reprogramming Medium supplemented with 10 µM Y27632 (2.1.6). After centrifugation at 200 x g for 2 min, the cell pellet was washed once with PBS to remove the FB medium completely. The cells were centrifuged at 200 x g for 2 min, resuspended in 4 ml NuFF-conditioned Pluriton™
Reprogramming Medium supplemented with 10 µM Y27632, seeded on two matrigel coated 6-wells (2.2.4.1.2) and further cultured at 37°C, 5% CO2 and 5% O2. The next day the transfection series was continued according to the plan (Tab. 1) and passaged cells were further cultured in NuFF-conditioned Pluriton™ Reprogramming Medium supplemented with 10 µM Y27632 till the end of reprogramming.
2.2.4.11.2.5 Picking and passaging of emerged hiPSC colonies
When hiPSC colonies exhibited their characteristic morphology with defined colony edges, they were picked on MEF feeder layer (2.2.4.2.1) in order to expand them and to establish hiPSC cell lines. During the working procedure a surgical mask was worn in order to minimize the risk of contamination. Cells were washed once with PBS and covered with appropriate medium. Observed under the light microscope, which was disinfected and put
under the opened sterile bench, hiPSC colonies were broken into 4-8 fragments and detached manually from the culture plate by using a needle. Here it was important to avoid single cell suspensions. Colony fragments were transferred in appropriate medium on freshly prepared MEF feeder layer using a serological pipette and placed in the incubator at 37°C, 5% CO2 and 5% O2, where the plate was gently rocked from side to side and front and back to distribute the hiPSC aggregates across the well. The first medium change was performed 48 h after picking of cells.
First hiPSC colonies were cultured in NuFF-conditioned Pluriton™ Medium (2.2.4.11.1.3) supplemented with bFGF (= 4 µl P™S in 10 ml medium) to a final concentration of 20 ng/ml and hiPSC-M3 mixed in a ratio of 50:50, which was reduced stepwise during further culture in order to reach 100% hiPSC-M3 (2.1.11.1). After culturing in pure hiPSC-M3 this medium was replaced with final hiPSC medium (2.1.11.1) in order to expose hiPSCs to a reduced bFGF concentration of 5 ng/ml, which decreases the risk of spontaneous differentiation of hiPSCs. Furthermore the culture of picked hiPSCs was stepwise propagated on 12-well, followed by the use of 6-well vessels.