Culture of eukaryotic cells

2.2.4.3.1 Culture of murine embryonic stem cells

In order to keep embryonic stem cells (ESCs) in their undifferentiated state, they were continuously cultured on mitotically inactivated mouse fibroblasts (MEF feeder layer;

2.2.4.2.1) and in ESC medium containing the cytokine LIF (Leukemia Inhibitory Factor;

2.1.11.1).

2.2.4.3.2 Culture of murine embryonic fibroblasts

Feeder cells are indispensable for the culture of ESCs. These are mitotically inactivated murine embryonic fibroblasts (MEFs), which are homogenously seeded on gelatine treated culture vessels and cultured in FB medium (2.1.11.1). A treatment with mitomycin C leads to the non dividing status of these cells (Martin and Evans, 1975).

2.2.4.3.3 Isolation of murine embryonic fibroblasts

Mouse embryos were isolated from gestating mice at stage 13.5-15.5 days post coitum (dpc) and washed in PBS. After opening of the abdomen and removal of organs, the heads were separated and the bodies were shredded. The cell suspension was transferred in a glass bulb with glass pellets and 20 ml Trypsin/EDTA and 1 ml DNAse were added. After 30 min incubation at 37°C with mixing, the cell suspension was centrifuged by 270 x g for 10 min.

The supernatant was discarded and then resuspended in FB medium. The cells were seeded in appropriate culture vessels. After approximately 24 h of cultivation the medium was changed to remove not adherent cells. After further cultivation of approximately 3 days, the cells were passaged in a ratio 1:3. The cells were then cryopreserved after 3-5 days in passage No. 1.

2.2.4.3.4 Passaging of eukaryotic cells

Depending on the cell line and cell density, the cells were cultured for 2 to 4 days until they reached confluence for passaging. The cells were washed with PBS and further treated with 1-5 ml trypsin/EDTA, depending on the size of the culture vessel. After 3-5 min at 37°C and 5% CO2, all cells were detached from the culture vessel, which was controlled unter the light microscope. An equal volume of appropriate culture medium was added to inactivate the trypsin/EDTA treatment. The cell suspension was centrifuged at 1000 x g for 5 min. After the supernatant was discarded, the cell pellet was resuspended in appropriate culture medium (2.1.11.1) and seeded on an appropriate number of culture vessels. After approximately 24 h of cultivation at 37°C and 5% CO₂, the medium was changed to remove not adherent cells.

2.2.4.3.5 Cryopreservation and thawing of eukaryotic cells

Cells were cryopreserved from a confluent culture vessel. After washing with PBS, the cells were treated with 1-5 ml trypsin/EDTA, depending on the size of the culture vessel, and

cultured for 3-5 min at 37°C and 5% CO₂. Under the light microscope, it was controlled that cells were detached from the culture vessel. The effect of the trypsin/EDTA treatment was inactivated by an equal volume of appropriate culture medium and the cell suspension was centrifuged at 1000 x g for 5 min. After the supernatant was discarded, the cell pellet was resuspended in 500 µl appropriate culture medium (2.1.11.1) and subsequently mixed with 500 µl appropriate freezing medium. The mixture was transferred into well labelled cryovials, which were immediately frozen at 80°C. After 24 h at 80°C, the cryovials were placed at -152°C for long term storage.

Because the DMSO contained in the freezing medium is toxic for cells at room temperature, the procedure of thawing has to be done quickly. The cryovial was placed in a water bath at 37°C until the cells were almost completely thawed. The cells were mixed with a bigger volume of appropriate culture medium in order to dilute the DMSO and then centrifuged at 1000 x g for 5 min. After removal of the supernatant, the cells were resuspended in appropriate culture medium and seeded on a culture vessel. After approximately 24 h of cultivation at 37°C and 5% CO₂, the medium was changed to remove not adherent cells.

2.2.4.3.6 Culture of Human Testicular Feeder

Human Testicular Feeder (HTF) cells were derived from MACSortings (2.2.4.7.2) with human testicular biopsies from infertile men. During this procedure the flow through was captured in an e-cup, which was stored on ice, if HTF cells had to be established. The cell suspension was centrifuged at 1000 x rpm for 5 min, resuspended in 2 ml FB medium, seeded on one 6-well vessel coated with 0.1% gelatine and cultured at 37°C and 5% CO₂. During further culture, cells were expanded, routinely passaged (2.2.4.3.4) and cryopreserved using FB freezing medium (2.2.4.3.5).

2.2.4.3.7 Digestion of human testicular material

Testicular biopsies were obtained simultaneously during surgery for testicular sperm extraction (TESE) for the diagnosis of male fertility from patients as previously described (Jezek et al., 1998; Schulze et al., 1999; Feig et al., 2007). TESE was performed by Prof.

Schulze at the Department of Andrology, University Hospital Hamburg-Eppendorf in Hamburg. Ethic Committee Approval to Prof. Schulze was obtained (OB/X/2000) and the study was performed according to the ethical guide lines of the current Declaration of Helsinki.

Testicular tissue was kept in medium at ~32°C during the transport to the Institute of Human Genetics in Göttingen. Upon arrival testicular biopsies underwent a two-step enzymatic digestion procedure. Testicular biopsy was placed in culture plates containing HBSS and

was washed in HBSS by using forceps. Further it was transferred into culture plates containing HBSS supplemented with 1 mg/ml collagenase and 2 U/ml DNaseI. Tissue was mechanically dissociated into small pieces by using forceps, transferred into a 15 ml falcon tube and digested for 15 min at 37°C. During the incubation time testicular pieces were occasionally mixed by shaking. After centrifugation at 1000 x rpm for 5 min, the supernatant was carefully removed and the pellet was resuspended in HBSS supplemented with 1 mg/ml collagenase, 2 U/ml DNaseI and 0.5 mg/ml hyaluronidase. The suspension was carefully mixed six times by pipetting using a glass pasteur pipette and digested for 10 min at 37°C. During the incubation time the tissue was occasionally mixed by shaking. After addition of 1 ml FCS to inactivate enzymes, the tissue solution was extensively mixed by pipetting using a glass pasteur pipette and transferred with a 10 ml serological pipettes on a nylon mesh cell strainer (40 µm pore size) to remove cell debris. The cell suspension was captured in a falcon tube, centrifuged at 1000 rpm for 5 min and the supernatant was discarded. The cell pellet was washed once in PBS and centrifuged at 1000 rpm for 5 min. For isolation of human spermatogonial stem cells these cells underwent a MACSorting (2.2.4.7.2).

2.2.4.3.8 Culture of Scrotal Human Fibroblasts

Scrotal Human Fibroblasts (SHF) were derived from scrotal skin from infertile men. Scrotum biopsies were obtained simultaneously during surgery for testicular sperm extraction (TESE) for the diagnosis of male fertility from patients at the Department of Andrology, University Hospital Hamburg-Eppendorf in Hamburg, embedded in medium containing vials and send by mail to the Institute of Human Genetics in Göttingen for further study. Ethic Committee Approval to Prof. Schulze was obtained (OB/X/2000) and the study was performed accordingl to the ethical guide lines of the current Declaration of Helsinki. On arrival scrotal skin samples were cultured on 0.1% gelatin-coated culture dishes in FB medium at 37°C and 5% CO2. During culture, the cells expanded from scrotal skin and started to proliferate.

These so called SHF cells were expanded, routinely passaged (2.2.4.3.4) and cryopreserved using FB freezing medium (2.2.4.3.5).

2.2.4.3.9 Co-culture of mouse ESCs with HTF cells

Mouse ESCs were cultured under standard ESC culture conditions at 37°C and 5% CO2. Using FB medium co-culture experiments were performed with 100.000 mESCs seeded on mitotically active HTF cells, which reached nearly 80% confluence. First medium change was performed 3 days at the earliest after starting the co-culture, subsequently every second to third day. After 10-12 days in co-culture cells were passaged in a ratio of 1:2-1:3 on 0.1%

gelatine-coated culture dishes and further cultured for at least 8-10 days. These cells were used for further analysis.

2.2.4.3.10 Cultivation and passaging of hiPSCs

Generally, established hiPSCs were cultured in hiPSC medium (2.1.11.1) on 6-well vessels providing a MEF feeder layer (2.2.4.2.1) at 37°C, 5% CO₂ and 5% O₂ (hypoxia). Every second day cells were washed once with PBS and covered with equilibrated hiPSC medium.

hiPSCs had to be passaged when colonies filled in the size observed through a 10x objective of the light microscope, colonies physically contacted each other and/or the MEF feeder layer was older than 7 days. Depending on the confluence, hiPSCs cultured on one 6-well vessel were passaged in a ratio of 1:2 – 1:4.

For passaging, hiPSCs were washed once with PBS and covered with 1 ml accutase for enzymatic cell detachment. Cell dissociation was observed under the light microscope. When the colonies started to dissociate from the culture well and the edges of colonies were detached, the accutase was carefully removed from the frame of the culture well and cells were immediately covered with equilibrated hiPSC medium. hiPSC colonies were collected by scratching the surface of the 6-well vessel with a serological pipette and simultaneously rinsing with medium. Remaining colonies were collected with a cell scraper. Collected cells were pelleted by gently centrifugation (300 x g, 5 min) and carefully resuspended in an appropriate volume of equilibrated hiPSC medium supplemented with 10 µM Y-27632 (2.1.6) without making a single cell suspension. According to the passaging ratio hiPSCs were seeded on one ore more 6-well vessels coated with MEF feeder layer and the 6-well plate was placed in the incubator at 37°C, 5% CO₂ and 5% O₂, where it 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 passaging procedure.

2.2.4.3.11 Cryopreservation and thawing of hiPSCs

The procedure for cryopreservation and thawing of hiPSCs had to be established in the presented thesis. The establishment was carried out by testing different ways of enzymatic cell detachment, cell collection, centrifugation, compositions of freezing media and freezing procedures, which is summarized in Tab. 7 in the corresponding result part (3.2.3).

The survival of hiPSC colonies cryopreserved by the tested procedures was evaluated by counting the number of emerged colonies after replating on MEF feeder layer. The presence of Rho associated kinase (ROCK) inhibitor Y-27632 is known to increase hiPSC colony formation (Watanabe et al., 2007) and to improve the recovery and further growth of cryopreserved hiPSCs by exhibiting anti-apoptotic activity (Claassen et al., 2009). Because

of these findings, thawed hiPSCs were always cultured in appropriate hiPSC medium supplemented with Y-27632 (10 µM) for 48 h after replating on MEF. Additionally, the impact of pre-incubation of hiPSCs with Y-27632 before cell detachment was tested. Cell detachment was performed with or without using cell dissociation. Enzymatic cell detachment was performed using collagenase or accutase, while cell dissociation was observed under the light microscope. Finally, hiPSC colonies were collected by using a needle, scratching with a serological pipette and cell scraper or rinsing with medium followed by scratching with a cell scraper. Collected cells were pelleted by gently centrifugation (200-500 x g, 3-5 min) and carefully resuspended in appropriated freezing medium. To asses the best freezing medium, hiPSCs were frozen in freezing media according to Wagner and Welch (2010), containing KO™-SR and DMSO (90:10) with or without Y-27632 as well as in commercial cryopreservation media (CryoStem™ Freezing Medium; Biofreeze and hiPSC medium (90:10) with Y-27632). For cryopreservation vials were frozen directly in -80°C or cooled -1°C/min (using Mr. Frosty™ Freezing Container) in -80°C. After 2-3 days stocks were transferred to -152°C for long term storage. Cryopreserved stocks were recovered by thawing frozen hiPSCs in a 37°C water bath until the vials’ content was almost, but not completely thawed, and diluted with at least 4 ml hiPSC medium in a 15 ml falcon tube. After centrifugation (200-500 x g, 3-5 min), the cell pellet was gently resuspended in appropriate hiPSC medium supplemented with Y-27632 without making a single cell suspension and finally repleated on MEF feeder layer. Different freezing/thawing methods led to the emergence of hiPSC colonies after cryopreservation. Nevertheless, enzymatic cell detachment using accutase revealed better efficiencies of colony formation. The survival rate of hiPSCs could be increased by using Y-27632 in freezing medium containing KO™-SR and DMSO (90:10). However, the best survival rate of cryopreserved hiPSCs was achieved using commercial CryoStem™ Freezing Medium, in which cells were frozen gradually -1°C/min in -80°C. In general, formation of small clusters with a compact morphology could be observed earliest three days after replating on MEF feeder layer. Final hiPSC colony formation with clear borders could take up to 14 days.

After testing these different conditions, hiPSCs were cryopreserved using freezing medium containing KO™-SR and DMSO (90:10) supplemented with 10 µm Y-27632 as well as using CryoStem™ Freezing Medium. For the purpose of cryopreservation, hiPSCs were pre-incubated with equilibrated hiPSC medium supplemented with 10 µM Y-27632 for 1 h at 37°C, 5% CO₂ and 5% O₂. Then hiPSCs were treated with accutase and pelleted according to the protocol for passaging of hiPSCs (2.2.4.3.10). After centrifugation the pellet was resuspended in either 1 ml ice cold CryoStem™ Freezing Medium and the cryopreservation vial was directly cooled -1°C/min in -80°C using Mr. Frosty™ Freezing Container or in 1 ml freezing medium containing KO™-SR and DMSO (90:10) supplemented with 10 µM Y-27632

and the cryopreservation vial was frozen directly in -80°C. After 2-3 days stocks were transferred to -152°C for long term storage.

For thawing of cryopreserved hiPSCs, the cryovial was placed in a water bath at 37°C until the cells were almost completely thawed. The cells were mixed with a bigger volume of hiPSC medium in order to dilute the freezing medium and then centrifuged at 300 x g for 5 min. After removal of the supernatant, the cells were carefully resuspended in an appropriate volume of equilibrated hiPSC medium supplemented with 10 µM Y-27632 and seeded on one 6-well vessel prepared with MEF feeder layer. The 6-well plate was placed in the incubator at 37°C, 5% CO₂ and 5% O₂, where it 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 thawing procedure.