Experiments in the culture of human germ cell lineages
Experiments in the culture of human germ cell lineages
This chapter focuses on embryonic germ cells (E G C ) and aims to provide a series of experimental protocols that can be utilized to understand the relationship between E G C and other human stem cells.
Author: Stacey et al., Translated by Jingbo Zhang, from "Human Stem Cell Cultures".
Operation method
Experiments in the culture of human germ cell lineages Move I. Rules for the Preparation of Culture Base and Reagents 1. Cellular Dissociation Maintenance Medium (CDM) (i) Hank's Balanced Salt Solution (HBSS) (ii ) IV Collagenase, 2. 5 mg/ml (iii) DNase I , 20U/ml (iv) heat-inactivated neonatal bovine serum, 2 % V/V (v) CaCl2, 0 -54 mmol/L (60ug/ml) (i) D M E M (ii) Fetal Bovine Serum (FBS), 2 % V/V (iii) Penicillin, lOOU/ml (iv) Streptomycin, 100ug/ml De-serumized D M E M contained: (i) de-serumized substitutes (KO-SR), or ESC test fetal bovine serum (ESC-FBS) ...... 1 5 % V/V (ii) L-glutamine ............................. lmmol/L (iii) 2-hydroxyethanol ......................... 0. lmmol/1 (iv) Non-essential amino acids IOOX ........................ ...1%v/v (V) Penicillin ........................... .... .100U/ml All cultures are carried out at 5% C02, 95% humidity, 37°C, and operated in a laminar flow hood (Class II microbiological safety cabinet). We clean surfaces, tubes, and utensils with 75% ethanol, and all cell culture plasticware is sterile. Various types of mouse and human embryonic cells have been shown to support the growth of PGCs and promote the derivation of hEGCs. However, in our experiments, we also found that a feeder cell line, STO fibroblasts, had a significant promotional effect, as did other scholars [Shamblott etal. 1998; 2004]. The following programs have been developed for Option 1 Preparation and culture of feeder cells Reagents and materials Sterile or aseptic preparation □ STO fibroblasts (ATCC CRL 1503; ECACC) □ feeder layer cell culture medium (F C M ; see section 5. 2. 2) □ feeder cell culture medium (FCM; see section 5.2.2) □ PBSA □ Trypsin, 0.25%, E D T A , lmmol/L, dissolved in PBSA □ culture flask, 75 cm2 or 150 cm2 □ Sterile conical-bottomed centrifuge tubes, 15 ml or 50 ml Steps (a) Resuscitate a tube of S T O fibroblasts from a liquid nitrogen tank and partially immerse in a 37° c water bath to promote rapid lysis. (b) When 90% of the cell suspension is lysed, transfer the frozen tube to a cell culture ultra-clean bench and clean the surface of the frozen tube with 75% ethanol. (c) Add pre-warmed FCM to the tube and transfer the cell suspension to a 15mi centrifuge tube containing 6m i of pre-warmed FCM. (d) Centrifuge at 250 g for 3 min (e) Discard the supernatant, resuspend the cell pellet with pre-warmed FCM, and transfer to a cell culture flask (culture volume: 10 ml/75 cm2 or 20 ml/150 cm2 , and put into the incubator for culturing feeder cells. (f) Change the medium regularly (every two days). (g) When the cells have grown to confluence or near confluence, aspirate the medium and wash gently with pre-warmed sterile P B S A . (h) Add pre-warmed trypsin-E D T A to the culture flasks to dislodge the cells at the time of passaging, 3 ml for 75 cm2 flasks and 6 ml for 150 cm2 flasks, ensuring that the entire culture surface is covered. Tap the flasks from time to time to dislodge the cell layer from the flask wall after about 3 min (it is not necessary to return the flasks to the incubator for this procedure). (i) Add pre-warmed FCM (3 ml of FCM for every Iml of trypsin) to terminate the activity of trypsin, and gently blow with a pipette repeatedly to dislodge the remaining adherent cells. (j) Transfer the cell suspension to a sterile 15 ml or 50 ml centrifuge tube and centrifuge at 250 g for 3 min to settle the cells. (k) Resuspend the cell sediment with IOml warm FC M and re-inoculate with 1:5 dilution. (l) Repeat the passaging step, which should yield about 20 to 30 vials (75 c m 2) of pooled STO fibroblasts at a time. Treatment with mitomycin C or irradiation with ionizing rays can stop cell growth. We have had good experience with the use of gamma rays. Our experimental protocols are laborious and require advance planning and access to appropriate instrumentation. However, it is possible to prepare a number of spare cells for freezing (one tube per 75 c m 2 of flask growth), which ensures that a homogeneous monolayer of cells can be obtained without the need for continuous preparation, cell counting (if the cells are not in the cell count), or for the cell counting (if the cells are not in the cell count). Scheme 2 Growth blockade of feeder layer cells Reagents and materials sterile Growing STO fibroblasts (see Option 5.1) feeder cell cultures (FCM; see Section 5.2.2) Feed layer cell freezing medium (FFDM; see section 5.2.4) P B S A □ Trypsin, 0.25%, E D T A, l m m o l /L , dissolved in P B S A □ culture bottle, 75 c m 2 or 150 c m 2 □ conical-bottomed centrifuge tube, 50 ml, sterile □ freezing tubes □ gamma sources (e.g., 137C s ) □ Controlled rate freezing vessel containing 250 ml of isopropyl alcohol Steps (a) Radiation treatment must be carried out when the cells have grown to confluence (approximately 8 X 106 cells per 75 cm2 flask), and our experience has been that it is best not to treat the cells after this point. (b ) Change the medium the night before irradiation. (c) Aspirate the medium and gently wash the cells with pre-warmed sterile P B S A . (d) Dislodge the cells by adding pre-warmed Trypsin-Edta to the culture flasks, 3 ml for 75 c m 2 flasks and 6 ml for 150 c m 2 flasks, ensuring that the entire culture surface is covered. Tap the bottle from time to time, and after about 3 m i n , the cell layer can be detached from the wall of the bottle (it is not necessary to return the bottle to the incubator for this procedure). (e) Terminate trypsin activity by adding pre-warmed FCM (3 ml of FCM per 1 ml of trypsin), and gently blow repeatedly with a pipette to dislodge the remaining adherent cells. (f) Transfer the cell suspension into sterile 50 ml centrifuge tubes (for ease of operation, the volume of liquid in each tube should not exceed 30 ml), and centrifuge at 250 g for 3 min to settle the cells. (h) The resuspended STO cells were deactivated mitotically by irradiation with 50 G y of γ-rays (we used a 37-MBq137C s source for 23 min to achieve this dose), which is appropriate for STO cells of this quality and density, and which may need to be adjusted for different cell lines or different cell numbers. (i) Centrifuge at 250 g for 3 min to settle the cells. (j) Resuspend the cells well with pre-warmed FFD M. Use l ml of cryopreservation solution for each 75 cm2 flask. (k) Maintain a homogeneous cell suspension, dispense 1 mI into individual cryotubes, and place in controlled rate freezing containers at 80°C overnight. (l) The following morning, transfer the tubes to a liquid nitrogen tank. For laboratory research, it is important to obtain human clinical material of a certain quality and within a specific time frame, and the use of human clinical material often cannot be planned in advance in the same way as the use of animal models. It is preferable to inoculate the growth-blocking monolayer fibroblast feeder layer the day before starting the human germ cell culture, but if inoculated a day earlier, the feeder cells are still available. When experimental material was temporarily available for sampling, we found that feeder cells could successfully adhere to the culture surface within 5 h. This is partly due to the fact that the medium used to inoculate feeder cells is different from that used to inoculate germ cells: the FCM contains serum, which contains factors that promote cell adhesion to the surface of the culture vessel, whereas the removal of serum substitutes in the GCM (KO-SR, see section 5.7) does not contain these "adhesion factors". ) in GCM does not contain these "adhesion factors". There are a number of uncertainties associated with the establishment of germ cell cultures (see section 5.3.4); it may take more than a week for cells to begin to grow visibly, whereas the average time for feeder cells to survive is about 10 days, which is why feeder cells should not be pre-inoculated for more than two days prior to performing a germ cell culture. Also, the conversion of FCM to GCM on the morning of the day the tissue is taken allows the GCM to be assimilated by factors released by the feeder cells. As mentioned in Scheme 5.2, radiation-treated feeder cells are frozen at a rate of one tube per 75 cm2 flask full of confluent cells, a number of cells that can be inoculated over a surface area equivalent to approximately one IOcm diameter petri dish. Thus one frozen tube contains enough cells to form a monolayer in an IOcrn diameter dish, or proportionally 3 to 4 6 cm diameter dishes, 8 3.5 cm diameter dishes, or one 6-well plate, 1 ¾ 12-well plates, and 1.5 24-well plates. The same can be inferred for 96-well plates or fibronectin pre-coated slides (Scheme 5.4). Scheme 3 Inoculation of growth-blocked monolayer feeder cells Reagents and materials Sterile or aseptic preparation S T O fibroblasts without dividing activity (see Scheme 5. 2) □ Word-foster layer cell culture medium (F C M ; see section 5, 2, 2) □ germ cell medium (GCM; see section 5. 2. 3) □ PBSA Trypsin, 0.25 %, lmmol/L EDTA, dissolved in PBSA 25 %, lmmol/L ED T A , dissolved in PBSA □ Petri dishes (6 or 3_5 cm diameter petri dishes or 24, 12, 6-well culture plates) □ Cone-bottomed centrifuge, 15 ml Steps (a) One day before the start of germ cell culture, remove a tube of radiation-treated STO fibroblasts from the liquid nitrogen tank and partially immerse them in a 37°C water bath to promote rapid lysis. (b) When 90% of the frozen material is lysed, transfer the frozen tube to a cell culture ultra-clean bench and clean the surface of the frozen tube with 75% ethanol. (c) Transfer the cell suspension to a 15m l centrifuge tube containing 6m l of pre-warmed FCM, mix by briefly blowing gently, and centrifuge at 250 g for 3m i n . (d) Resuspend the cells with F C M by adding one-half of the total culture volume; add the other half of the volume of medium to the culture vessels or culture wells; add the resuspended feeder cells to the culture vessels or culture wells proportionally and equally. Carefully place these culture vessels in a cell culture incubator. (e) The following morning (i.e., the day the tissue was taken), aspirate the FCM and gently wash the cells with sterile P B SA. (f) Remove the PBSA and gently add pre-warmed GCM to reach half the total culture volume of germ cells (see Protocol 5.5). (g) Return the culture vessel to the incubator until germ cells are inoculated. We inoculate plastic tissue culture vessels with a feeder layer of irradiated fibroblasts, followed by inoculation of human gonadal cells, or we prepare slides for cell culture and inoculate the cells; glass culture slides with small chambers are suitable for experiments in which fluorescence immunocytochemistry is subsequently used to analyze the cell type (Option 5.8). For effective attachment of fibroblasts, the slides should be coated with some extracellular matrix mimic before use. Fibronectin has been found to be the cheapest and most reliable pro-adhesion factor, although its principle is not different from that of other reagents such as laminin or Matrigel. Similarly, gelatin-coated Petri dishes can be used to study E G C differentiation. Option 4 Gelatin-coated slides or tissue culture dishes with culture chambers Reagents and materials sterile □ gelatin, 2 % sterile solution □ Fibrinogen (bovine; l m g/U) □ slides with chambers Steps (a) Remove the slide with chambers from the sterile package in the cell culture ultra-clean bench. (b) Aspirate fibronectin into the center of each chamber at 5ug/cm 2 and form an air bubble by expelling the air from the pipette to spread the fibronectin over the entire surface of the well. (c) Cover the slides with the chambers and dry them in a tissue culture ultra-clean bench. In our experience, prepared slides can be stored at 4°C for at least one week. (d) If the petri dish is to be gelatin-coated, apply a 0.1% gelatin working solution in sterile water to the surface of the dish, let it stand for I h, then aspirate the liquid, leave the lid open, and place the dish in a tissue culture ultra-clean bench to dry. Gelatin-coated Petri dishes can be stored at 4°C for at least one month. In the UK, the use of human embryos and fetal material is governed by the Code of Practice established by the PoIkinghorne Committee in 1989 [PoIkinghorne, 1989]. In addition, ethical approval is required for research projects and material can only be taken after informed consent has been signed with women who are prepared to terminate an abortion in the community in which they are pregnant (or voluntarily). Our experiments have removed the germinal cristae from embryos approximately 7 to 11 weeks after conception (w p c ), identified and descaled under a dissecting microscope with sterile instruments and containers. This manipulation minimizes the risk of subsequent contamination. However, it is important to recognize that material obtained transvaginally is not sterile, so we use a dedicated incubator to keep primary human germ cell cultures separate from all other cultures. The isolated germinal cristae are placed in sterile H B S S and transferred to an ultra-clean bench for cell separation. We describe two methods of dissociation, both of which are variable, presumably with a number of uncontrollable factors, including : the different developmental stages at which the material is located; the time between taking the material and starting the operation; and the number of samples available for manipulation. The final cell suspension obtained will contain individual cells, small clusters of cells, and large undissociated clumps of cells, which will settle, and the individual cells and small clusters of cells will be pipetted out for inoculation. Although the first method may yield a larger number of cells, a protease enzyme is used to lyse a certain percentage of the cells, producing potential damage to important proteins on the cell surface. In contrast, the second method may yield a smaller number of cells, but avoids prolonged washing and resuspension, minimizing potential damage. We have found that both methods can be effectively applied to the initial h E G C culture, however to date we have not been able to determine which method yields more. Similarly, we have determined that there is little difference in the cultures obtained by retaining or removing mesonephridia from the germinal crest. Our germ cell cultures are listed in section 5. 2. 3 The question that remains unclear to us is whether the addition of forskolin, recombinant human fibroblast growth factor 2 (F G F -2), and recombinant human leukemia inhibitory factor (L I F ) makes a significant difference in establishing and maintaining germ cell cultures. The published literature on h E G C culture, and also our article, adds f this search factor [Shamblott et al. 1998; T u r n p e n n y et a L ,2003; L i u e t a L , 2004: P a r k e t a l . , 2004] OPTION 5 Anatomical isolation and cellular inoculation of human embryonic gonads Reagents and materials Sterilized or aseptically prepared □ pre-inoculated growth-blocked STO fibroblasts (see Option 5.3) □ Reproductive cell culture medium (GCM; see section 5.2.3) H an kS balanced salt solution (HBSS) □ EDTA, disodium salt, 0.3 mmol/L (0.01%), soluble in PBSA □ Cell Dissociation Medium (CDM, see section 5. 2. 1) □ Conical bottom centrifuge tube, 15 ml Scalpel □ Needle, 18 G □ glass petri dish or slide for dissection □ Nylon mesh, IOOum pore size, cut according to the size of petri dishes, autoclaved. □ Shake table with temperature set at 37°C and speed of approximately 200r/m i n . Steps (a) Dissect out the gonads (with or without the mesonephros) and place them in a sterile H B S S (see Cabinet 5.2). (b) Prepare cells for inoculation by any one of the following methods. Procedure 1 ① Soak the tissue in EDTA solution for IO m in and return it to HBSS. ② On a sterile glass dish (or slide), mechanically dissociate the tissue with a sterile scalpel and forceps, keeping the tissue in a small amount of HBSS to avoid drying out. ③ Transfer the material to a 15m l cone-bottomed centrifuge tube containing 2m l of Cell Dissociation Solution (C D M ) , shake at 37°C for 1~2 h on a shaker, and grind randomly with a syringe with an 18 G needle. ④ Centrifuge at 250 g for 3 m i n . ⑤ Carefully aspirate the supernatant and wash at will. If this step is not done, the precipitate obtained after centrifugation in step 4 can be resuspended directly (step 7). (6) Add 3 ml of HBSS and gently resuspend the cell pellet by blowing with a pipette, then centrifuge again at 25 s.c. for 3 min and carefully aspirate the supernatant. (vii) Resuspend the cell pellet with pre-warmed GCM (half of the total culture volume, refer to Option 5.3, step 7), and let it stand for 3 to 5 m i n . Procedure 2 The tissue was immersed in ED TA solution for 20 m in and then returned to HBSS. ② Place the tissue in a sterile glass petri dish with a small amount of pre-warmed G C M . ③ Puncture and squeeze the entire organ under a dissecting microscope with a sterile dissecting needle and forceps to release the cells directly into the culture medium. ④ Transfer the medium to a 15 ml pointed-bottom centrifuge tube containing pre-warmed G C M [keep half of the entire culture volume, refer to step (f) in the previous scheme 5. 3 ] , and leave the cell suspension for 3 to 5 m i n . (c) Collect single cells or small clumps of cells from the supernatant by pipette for inoculation, or optionally, filter the cell suspension through a sterile, IOOum-size nylon mesh (moisten the underside of the mesh with culture medium and add the suspension to the top of the mesh). (d ) Gently dispense the cells from one side to the other into selected dishes or multi-well plates that have been pre-inoculated with feeder layer cells, and add the cells directly to the culture medium using a pipette. (e) Place the culture vessels in a specialized CO2 incubator. Our experience is that antibiotics should be added to all primary cultures of isolated gonadal-derived cells, at least when using initial material from non-sterile sources, but there is no need for antifungal reagents. However, mycoplasma infections are readily present, so initial and maintenance cultures are placed in a dedicated incubator. Once the culture is established, routine testing for mycoplasma is performed as evidence, along with nuclear characterization (see Option 5.9). We have used a variety of commercially available methods with no particular improvements. Scheme 6 Maintenance and Transmission of Gonadal Cell Cultures Reagents and Materials Sterile materials Reproductive cell culture medium (GCM; see section 5.2.3) □ sterile PBSA □ Trypsin, 0.25%, E D T A, l m m o l /L, dissolved in PBSA □ pre-inoculated non-dividing STO fibroblasts (see Option 5.3) □ Feeder cell culture medium (FCM, see section 5.2.3) □ Cell culture jars (diameter or 3 cm Petri dishes or 24, 12, 6-well plates) □ conical-bottomed centrifuge tubes. Steps (a) On the 3rd day of incubation, remove the culture from the incubator and decide on one of the following protocols depending on the turbidity of the medium. (i) If the culture has died of contamination, discard it. (ii) If the turbidity of the medium is due to the presence of large amounts of dead cell debris, replace the medium entirely. (iii) If the medium is moderately clear, replace half of the medium. (b ) Carefully aspirate the medium (completely or partially) and gently add fresh pre-warmed G C M . (c ) Repeat steps (a) and (b ) daily thereafter. (d) At week 2 of culture, identify and evaluate the cultures (see Option 5.4), and perform the first passaging of the selected cultures as follows. (e) Carefully aspirate the medium and gently wash the cells with pre-warmed sterile P B S A . (f) Add the smallest volume of pre-warmed trypsin-ED TA that only covers the cell growth surface (e.g., I m l for a 6 cm diameter dish) and return the culture vessel to the warmer. (g) After about 3 m i n , place the petri dish under a microscope and observe to see if the cells are detaching from the wall of the bottle. Once the cells start to detach, do not immerse the cells in the trypsin solution for a long time. (h) Add 5 m l of pre-warmed G C M to the culture vessel to inactivate the trypsin, gently blow to collect any remaining adherent cells, and, if desired, gently scrape the more firmly adherent colonies with a pipette tip or sterile cell scraper. If only a small amount of trypsin has been used, you may also choose to inactivate the trypsin by adding G C M in a volume of half the total amount of medium to be used subsequently, instead of 5 m l as described above, and proceed to step (1). (i) Transfer (collect as much as possible) the cells into a 15 ml conical-bottom centrifuge tube. (j) Centrifuge at 250 g for 3 m i n and discard the supernatant. (k) Gently resuspend the cell mass with half of the total amount of medium to be used subsequently. (l) Remove the culture vessel pre-inoculated with feeder cells from the incubator and gently place the cells into the chosen dish or culture plate (to be added drop by drop, so as to avoid damaging the cells as much as possible), avoiding damage to the feeder cell layer, and place the culture vessel back into the incubator. (m) Start from step (a) again for the next passaging. One of the major obstacles facing human germ cell cultures in our experiments, as well as those of others, is the difficulty of reliably evaluating the state of the cultures by observing the morphology of the colonies under a light microscope (Fig. 5 . 2 , Color Figure 5), and although colonies are visible, not all are located in the upper layer of the leptotrophic cells; some are nested in the space between the fibroblasts, where the extracellular matrix secreted by the radiation-treated cells may be present. On the other hand, only after fixation and testing can it be confirmed whether the cells that make up the colony are alkaline phosphatase (A P ) positive. This is different from what we do with h E S C s or h E C C s cultures, which have a more predictable colony morphology (see Fig. 5.3). To solve this problem, we usually use multiwell plates at primary culture and at passaging, so that some wells can be readily sacrificed for identification of germ cells to evaluate their growth without disturbing the growth of the bulk of the culture. Similarly, samples of resuspended cells at passaging can be removed for additional characterization (see section 5. 4.2). These steps already enable us to quickly predict which cultures should be taken into action with effort. During the first 2 weeks (usually at the first passaging), we evaluate the growth characteristics of the culture, its morphology, and the most critical indicator: the presence of alkaline phosphatase-active cells (see Scheme 5. 7). Based on these criteria, cultures can be classified into two types: "pp" (weakly proliferative) or "V p" (strongly proliferative). We characterize "VP" cells as h E G C status and have previously reported that approximately 15 % of primary cultures have been shown to be derivatized after this step [Dinh urnp en n y et a L , 2003]. Weakly proliferative cultures - "P P" It has been shown that these cultures contain only a limited number of germ cells, and A P staining reveals that only a few cells or a very small group of cells are positive, with a very low rate of colony formation (Fig. 5.4). Similar results were obtained by immunocytochemical staining of fixed cells with pluripotent markers. Interestingly, these cells survived for 50 days or more, and it was very difficult to convert them to a proliferative phenotype despite the continued addition of various factors (e.g., FGF-2), the trial of different combinations of media formulations, and a variety of surface substances that favored the cultures. Strongly Proliferative Cultures - "V P" These cultures form diverse colonies of various morphologies or networks of proliferative "migratory-like" cells that grow on resting feeder cell layers. A high percentage (>90%) of the cells are strongly positive for AP (see Figure 5.4). Colony growth and network proliferation persisted during passaging, as did immunoreactive activity for A P expression and other markers of pluripotent cells (see Schemes 5. 7, 5. 8). 8 ) of immunoreactive activity were also maintained. Network cells in V P cultures had a strikingly similar proliferative capacity to migratory P G C s in vivo compared with individual colonies. If the VP state is suggestive of a P G C to E G C transition, then, in contrast, the pP cultures indicate a failure to enter this state. Since P P cells are always present, these findings suggest that cell survival and the culture conditions that support it play little role in the ill-defined "conversion" or "derivation" of EGC cells. To date, little is known about the factors that determine h E G C s transformation. Characterization of human germ cells in culture can be done in parallel with control material, which can be either tissue sections or RNA extracts derived from the gonadal primordium at the same time, or hESCs or hECCs cultures. We were able to confirm the self-renewal of h E G C s in V P cultures by testing for several key markers, such as O C T s , S S E A s , and h T E R T . Similarly, the absence of such gene expression profiles is accompanied by the presence of differentiation markers , and by collecting transcripts and proteins it is possible to identify cellular lineages from the ectoderm, endoderm, and mesoderm. These identification methods meet the requirements for pluripotency in vitro and have been adopted by all research groups working on h E G C S culture [Shamblott et al. 1998; T u r n p e n n y et al. 2003; Liu et al. 2004; Park et al. 2004]. Alkaline phosphatase-activated coloration Detection of alkaline phosphatase activity is a reliable, readily available, and rapid means of monitoring germ cell profiles in human gonad cultures. In addition to the reagents specifically listed in Scheme 7, different commercial reagents are available. OPTION 7 Fixation and alkaline phosphatase staining of germ cell cultures Reagents and Materials Non-sterile materials □ PBSA □ polyformaldehyde (PFA), 4 %. □ Ethanol: 50%, 70%, 100 %, V/V □ Determination of buffer (see section 5.2.7) □ Nitro-blue tetrazolium chloride (NBT), 100 mg/ml 5-bromo-4-chloro-3'-indolyl phosphate p-p-toluidine (BCIP), 50 mg/ml □ Aquamount □ Deionized water Steps (a) Aspirate the medium from the culture vessel and wash the adherent cells gently with P B S A . (b) Aspirate off PB SA and add 4% PFA prepared with PBSA and fix for 3 min. (c) Aspirate off the fixative and wash with PB SA. If the sample is to be stored, follow steps (d) and (e). If the procedure is to be continued immediately, equilibrate briefly with assay buffer and proceed directly to step (g). (d) To remove the PBSA, dehydrate the cells by increasing the concentration of ethanol: 50 %, 70 %, 100 %, 2 min per step. (e) The culture vessels and fixed cells were allowed to dry at room temperature and stored at 4°C. The cells were then dried and stored at 4°C. (f) Incubate the surface of the culture with assay buffer for 2 min to rehydrate the cells. (g) Aspirate off the For more product details, please visit Aladdin Scientific website.
is to use such cells. S T O is an immortalized S I M (Sandoz Inbred mice) embryonic fibroblast cell line, which expresses murine membrane-bound stem cell growth factor, which is a germ cell discriminator in vivo as well as in vitro [ T u r n p e n n y e t a L , 2006]. However, as with any of the infinite cell lines, the properties of STO cells change between sublines over time in culture. Therefore, for consistency after a limited number of passages of cells from a single cryotube source, it is desirable to prepare a frozen stock of growth-arrested cells for preservation. Such cell sources are also available from suppliers such as the A T C C _ L G C Promochen joint venture or the European Cell Culture Conservation Center (ECACC). If there is a decrease in the support for the growth of PGCs, this batch of cells should be discarded and new cells should be prepared.
If mitomycin C treatment is used, counting is necessary). The cell counts in Option 2 are intentionally high because of the inevitable cell death during irradiation and cell freezing and thawing. In conclusion, based on our experience, this protocol saves time, saves consumables, and provides a stable monolayer of STO fibroblast feeder cells.
(g) Resuspend the cell suspension with IOrnl warm FC M and seal the tube.
Non-sterile
