Development and characterization of islet‐derived mesenchymal stem cells from clinical grade neonatal porcine cryopreserved islets

Porcine tissues display a great potential as donor tissues in xenotransplantation, including cell therapy. Cryopreserving clinical grade porcine tissue and using it as a source for establishing therapeutic cells should be advantageous for transportation and scheduled manufacturing of MSCs. Of note, we previously performed encapsulated porcine islet transplantation for the treatment of unstable type 1 diabetes mellitus in the clinical setting. It has been reported that co‐transplantation of islets and Mesenchymal stem cells (MSCs) enhanced efficacy. We assume that co‐transplantation of porcine islets and porcine islet‐derived MSCs could improve the efficacy of clinical islet xenotransplantation.


INTRODUCTION
7][8] In addition to their therapeutic effects, MSCs enhance the therapeutic effects of target cells when co-cultured or co-transplanted, [9][10][11] Further research into their therapeutic applications is underway.The demand for MSCs as sources of cell therapy is expected to increase in the future because, unlike embryonic stem cells, they do not pose ethical concerns and have fewer risks associated with gene transfer than induced pluripotent stem cells.However, securing sources for therapeutic applications of MSCs presents a challenge.Cell banks are necessary for the expansion of MSC therapy; however, securing sufficient allograft donors is difficult. 12Using autologous MSCs from a patient is also possible; however, there are safety issues associated with the collection of peripheral blood, bone marrow, 13 and adipose tissue. 14gs are physiologically and anatomically similar to humans.Furthermore, a stable supply of pigs is available.Recently, organ transplants from pigs to humans have been performed and medical use of pigs is expected to increase. 15,16By the way, controlling the risk of infectious diseases between humans and animals is also important for the medical use of pigs.Therefore, we focused on clinical-grade pigs with controlled infection risk as a source of MSCs for clinical applications.Clinical-grade pigs are designed pathogen-free pigs that have been created, assessed, and maintained in an isolated barrier facility as mentioned when discussing xenotransplantation regulations. 17eviously, we isolated and cultured mesenchymal stem cells from clinical grade neonatal porcine bone marrow (npBM-MSCs) and characterized them. 18Furthermore, we have confirmed the therapeutic efficacy of porcine-derived MSCs by transplanting npBM-MSCs into mice models of ischemic limb disease and diabetic wound healing. 19,20e therapeutic application of these cells are being investigated.
MSCs can be produced from various tissues within the body, and they differ from the derived tissues in their proliferative and secretory characteristics. 21Understanding the differences in the characteristics of MSCs depending on the derived tissue is crucial to determine the therapeutic effects of MSCs and their influence on co-culture with other cells.
Several studies have examined the use of MSCs in diabetes treatment, including bone marrow-derived MSC co-transplantation. 10Of note, we previously performed encapsulated porcine islet transplantation for the treatment of unstable type 1 diabetes mellitus in the clinical setting. 22,23We assume that co-transplantation of porcine islets and porcine islet-derived MSCs could improve the efficacy of clinical islet xenotransplantation.Furthermore, since we are able to create clinically available porcine islets, MSCs derived from the clinically applicable islets have advantages for regulatory approval.On the other hand, for regulatory approval, it is required to provide standard characterization of MSCs; therefore, in this study, we characterized our islet-derived MSCs.
To establish MSCs from tissue, including islets, the adhesion of the MSCs to the culture dish is an essential step, which requires several days of labor.By establishing MSCs from cryopreserved tissue, the procedure can be performed at any time, resulting in increased convenience.Although there are many studies on islet cryopreservation, most of them focus on evaluating the functionality of cryopreserved islets for transplantation [24][25][26] and there is limited information on the use of cryopreserved islets as a source of MSCs.
In this study, we investigated the establishment and characterization of MSCs from cryopreserved clinical grade neonatal porcine islets, which may serve as a source of therapeutic MSCs.

Animals
The study was approved by the Committee for the Care and Use of Laboratory Animals of Otsuka Pharmaceutical Factory, Inc. (Naruto, Japan).Clinical grade neonatal pigs were provided by Spring Point Project (New Richmond WI, USA), and non-clinical grade neonatal pigs were obtained from Kadoi Ltd (Ibaraki, Japan) (Table 1 ).The pigs were euthanized by bleeding from the abdominal aorta under anesthesia conditions and their pancreases were procured using a sterile technique. 27Pancreas were preserved using ice cold ET-Kyoto solution (ETK, Otsuka Pharmaceutical Factory, Inc.

Cryopreservation of islets of neonatal pigs
The islets were collected at modified RPMI in a 50-mL tube.The islet suspension was static, the islets were sedimented, and the supernatant was removed.The isolated islets were resuspended at 6000 IEQ/mL with Cellstor-S (lactated Ringer's solution with 3% trehalose and 5% dextran 40) (Otsuka Pharmaceutical Factory, Inc. Tokushima, Japan) containing 10% Dimethyl Sulfoxide (DMSO).Each cryovial contained 1 mL of isolated islets.The cryovials were placed in a BICELL (Nihon Freezer Co., Ltd., Tokyo, Japan) and frozen at −80 • C for 24 h before being transferred to liquid nitrogen tanks for long-term storage.

Thawing and culturing of islets to create npISLET-MSCs
The islets (6000 IEQ) in a cryovial were rapidly thawed in a 37 • C water bath, and 30 mL of MSC culture medium was gently added to the thawed cell suspension.Subsequently, it was centrifuged for 1 min at 210 × g at approximately 22 • C. The supernatant was removed, and 8 mL of MSC culture medium was gently added to the pellet.The MEM α (12571-063, Gibco) containing 10% FBS, 100 U/mL penicillin, 100 μg/mL streptomycin, and 1 ng/mL human fibroblast growth factorbasic (hBFGF, F0291, Sigma-Aldrich).The 6-well plate was placed in a CO 2 incubator and incubated at 37 • C, 5% CO 2 , and 90% humidity.Clinical grade npISLET-MSCs and non-clinical grade npISLET-MSCs reached approximately 50%-80% confluence after 3 days.Clinicalgrade npISLET-MSCs were established from islets cryopreserved for more than 4 years (Figure 1).

Passage of npISLET-MSCs
The cells from the 6-well plates were collected and replated into 75 cm 2 flasks after reaching approximately 50%-70% confluence.We washed the cells with 2 mL Phosphate buffered saline without cal-

Cryopreservation of npISLET-MSCs
If the npISLET-MSCs created are not used immediately, they are cryopreserved for future use.We washed the cells with 8 mL PBS (-) and for 24 h and then transferred to a liquid nitrogen tank for long-term storage.

Thawing and culturing of npISLET-MSCs
The npISLET-MSCs in a cryovial was rapidly thawed in a 37

Preparation, Thaw, culture, and passage of npBM-MSCs
The preparation, thaw, culture, and passage of npBM-MSCs were performed according to our previous study. 18

Cell viability
Cell viability was determined manually with a plastic cell counting plate (OneCell Counter, Fine Plus International Ltd., Kyoto, Japan) under a microscope by trypan blue staining.Cell viability was calculated according to the formula below: Cell viability [%] = (Total cell count − dead cell count) ∕ (Total cell count) × 100.

Cell proliferation assay
Cryopreserved cells were used after one or more passages.To plot the growth curve, passage-5 cells of non-clinical grade npBM-MSC and passage-3 cells of non-clinical and clinical grade npISLET-MSCs were plated at a density of 5000 cells/cm 2 (1.25 × 10 5 cells/flask) in a 25 cm 2 flask.The MSC culture media were changed every 3 days.
The same flasks were trypsinized, and the total number of cells was counted at 1, 2, 4, and 8 days.The growth curve was then plotted using the cell counting data.The doubling time (DT) was calculated using the following formula: where T is the time (h), N is the cell count, and T 0 , N 0 is the initial value.

Analysis of cell size
Analysis of cell size was performed at the end of the passage using a NuceloCounter NC-202.

Quantification of cell surface marker
To examine the cell surface immunophenotypes and forward scatter,

Adipogenic and osteogenic differentiation
Cryopreserved cells were used after one or more passages.We induced the adipogenic and osteogenic differentiation of clinical grade npISLET-MSCs.Induction of adipogenic differentiation was performed according to Poietics human mesenchymal stem cells -Adipogenic assay procedure (Lonza Walkersville, Inc.), and evaluated using Oil Red staining.Induction of osteogenic differentiation was performed according to the procedure manual of Poietics human mesenchymal stem cells -Osteogenic assay procedure (Lonza Walkersville, Inc.) and evaluated using Alkaline Phosphatase Staining kit (AK20, Cosmo Bio Co., Ltd.).

Colony forming unit (CFU) assay
Cryopreserved cells were used after one or more passages.Cells were plated at a density of 315 cells in a 21 cm 2 culture dish (30 cells/cm 2 ).
The MSC culture media were changed at day 3 and 6.After culturing for 8 days, adherent cells were washed twice with 4 mL PBS and fixed with

Analysis and statistics
Data are presented as the mean ± standard deviation (SD).Statistical analyses were performed using two-tailed tests, consisting of either Tukey's test or a non-paired Student's t-test, and a significance level of less than 5% was considered statistically significant.Data were analyzed using SAS 9.4 (SAS Institute, Inc., Cary, NC, USA).

Differences to establish neonatal porcine islet-derived MSC with and without cryopreservation
Fresh and cryopreserved non-clinical grade neonatal porcine islets were treated with Cellstor-S containing 10% DMSO for 5 days to prepare MSCs.The fresh islets were seeded in 6-well plates, and incubated at 37 • C, 5% CO 2 , and 90% humidity.Cryopreserved islets were rapidly thawed, washed with medium, seeded in 6-well plates and incubated at 37 • C, 5% CO 2 , and 90% humidity.Fresh islets and cryopreserved islets are both suspended in an MSC culture medium and seeded, resulting in cell growth (Figure 2A).It was found that both groups had a high level of positive expression of MSC surface markers such as CD29, CD44, and CD90 (Figure 2B).

Comparison of growth kinetics of non-clinical grade npBM-MSC, non-clinical grade npISLET-MSC, and clinical grade npISLET-MSC
MSCs were seeded at 5000 cells/cm 2 (1.25 × 10 5 cells/flask) in a 25 cm 2 flask, and both cells reached approximately 80% confluence on day 4 and 100% confluence on day 6.The calibrated growth curve was plotted for the non-clinical grade npBM-MSCs, non-clinical grade npISLET-MSCs, and clinical grade npISLET-MSCs (Figure 3A).

Immunological surface phenotypes of non-clinical grade npBM-MSC, non-clinical grade npISLET-MSC, and clinical grade npISLET-MSC
In all groups, positive MSC surface markers, such as CD29, CD44, and CD90, were highly expressed (Figure 5A).Negative surface markers, such as the endothelial marker CD31 and hematopoietic marker SLA-DR (a marker of swine corresponding to major histocompatibility antigen HLA-DR of human cells), were absent in all groups (Figure 5B).Hematopoietic marker CD34 of MSC negative marker was expressed slightly (9.1%, 5.8%, and 6.0%) in non-clinical grade npBM-MSC, non-clinical grade npISLET-MSC, and clinical grade npISLET-MSC, respectively (Figure 5B).

Adipogenesis and osteogenesis of clinical grade npISLET-MSC
The Oil Red stain (Figure 6A) and alkaline phosphatase stain (Figure 6B) were both positive.Therefore, our clinical-grade npISLET-MSCs differentiated into adipocytes and osteocytes.

CFU assay of non-clinical grade npBM-MSC and clinical grade npISLET-MSC
Colony-forming efficiency of cells of non-clinical grade npBM-MSCs and clinical grade npISLET-MSCs was 17.6 ± 2.8% and 21.7 ± F I G U R E 2 Continued 2.5%, respectively.Furthermore, there were no significant differences between the two groups.(Figure 7).

DISCUSSION
In addition to bone marrow 30 and fat, 31 various other tissues, including pancreatic islets, 32 can be used to establish MSCs.In this study, we demonstrate for the first time that MSCs can be established from islets cryopreserved for a period of more than 4 years.Islets are difficult to freeze, and after thawing, their functions such as viability and insulin secretion are reduced. 24Recent studies have shown that islets can be frozen and thawed by improving cryoprotectants and cryo methods. 25,26Generally, islets are not cryopreserved in clinical protocols to avoid loss of function. 33,34In contrast, MSCs are relatively resistant to freezing injury and have minimal loss of function and viability after thawing. 35Despite the possibility that the cryopreserved islets used in this study may have impaired insulin secretion and other functions, we speculate that this had an insignificant effect on the stem cells included in this study and thus, did not adversely To plot the growth curve, cells from passages 3 to 5 that were cultured in a 75 cm 2 flask without a gelatin coat were plated at a density of 5000 cells/cm 2 (1.25 × 10 5 cells/flask) in a 25 cm 2 flask without gelatin coat.The corresponding flasks were trypsinized, and the total number of viable and dead cells after 1, 2, 4, 6, and 8 days was counted.The growth curve was then plotted using the cell counting data.The DT was calculated from total cell counts immediately and 48 h after inoculation.Results are shown as mean ± SD values (n = 4).A: Different letters indicate significant differences by Tukey's test (p < 0.05).B: *p < 0.05, **p < 0.01.
affect the establishment of MSCs.In this study, MSCs were established from islets using a more general and simple method of slow freezing and thawing in a warm bath at 37 • C.Although pig islets have a fragile constitution, 36 we have demonstrated that they can be used to establish MSCs at any time, even after being frozen for more than 4 years.3][14] However, manufacturing under Good Manufacturing Practice (GMP) conditions is required.Clinical-grade npISLET-MSCs also need to be investigated using GMP-compliant materials in the future, as is being done for the GMP manufacturing of human islet MSCs. 37It is also known that various factors in the production of MSCs, such as source tissue, isolation protocols, media composition, and pre-treatment, affect the chemokine expression, immunomodulatory capacity, and therapeutic efficacy of MSCs. 38For npISLET-MSCs, there is insufficient information about secreted products, chemokines, and other characteristics that affect their therapeutic effect.There may be some characteristics related to their being derived from neonatal pigs rather than adults pigs, as observed in the bone marrow. 18In the future, these characteristics should be clarified, and appropriate quality control parameters should also be established.Furthermore, MSCs are known to change their proliferation rate, surface markers, and differentiation potential with passaging. 39Once quality control parameters have been determined, changes in character after passaging should be observed for npISLET-MSCs.We believe that use of pigs can allow production of MSCs on a larger scale than humans because there do not exist the same limitations associated with securing donors.It is important to establish MSCs with the same properties even from different batches in large-scale production.We have also isolated islets from another individual clinical-grade pig and established MSCs.A detailed study (A) F I G U R E 5 Immunophenotypes on the cell surface of MSCs as analyzed using flow cytometry.All cell types are prominently positive for CD29, CD44, and CD90 (A) and negative for the endothelial marker CD31 and hematopoietic markers SLA-DR.Additionally, the cells are weakly positive for the hematopoietic marker CD34 (B).Data obtained from a representative example (n = 1) are shown.
will be conducted in the future, but at least the growth curve of cell proliferation similar to that of npISLET-MSCs, but not BM-MSCs (data not shown).As mentioned earlier, it is necessary to maintain as stable a source of MSCs as possible when establishing production protocols and quality control parameters or clinical applications.In this regard, it is notable that the present study showed that porcine islets, the source of MSCs, can be cryopreserved for longer than 4 years, and that the established MSCs can be grown in sufficient quantities.
When MSC surface markers were measured, positive MSC surface markers such as CD29, CD44, and CD90 were highly expressed in all groups, and negative markers such as CD31 and SLA-DR were not seen.
CD34, a marker for hematopoietic stem cells, was weakly detected in all groups.However, it has also been reported that various progenitor cells, including MSCs, may show CD34 positivity. 40The npISLET-MSCs were established from islets after the purification process.Thus, we consider it unlikely that the MSCs were contaminated with hematopoietic stem cells.Clinical grade npISLET-MSCs were positive for CD44 and CD90 staining, but they showed a wider distribution than the other groups.Clinical grade npISLET-MSCs may be heterogeneous, but the number of studies examining this is small at this time, and data should be accumulated in future studies.
In the present study, the differentiation potential of clinical grade npISLET-MSCs was evaluated using a simple staining kit.Alkaline phosphatase staining has been used as a marker for osteoblast differentiation. 41,42In addition, alkaline phosphatase staining is also used to detect pluripotent stem cells. 43Future studies will need to evaluate osteogenesis in combination with other parameters such as osteocalcin antibodies and osterix mRNA expression level analysis.
Clinical-grade npISLET-MSCs showed positive oil red staining after induction of adipogenesis.However, a population of MSCs that maintained the morphology of MSCs rather than adipose tissue was also observed.Comparing the differentiation potential of pancreatic and bone marrow-derived MSCs, it has been reported that pancreaticderived MSCs are less adipogenic than bone marrow-derived MSCs. 44fferentiation data for clinical grade npISLET-MSCs are not yet sufficiently abundant.Therefore, it will be necessary to accumulate more data in the future.
MSCs are known to have different characteristics depending on their derived tissue.For example, adipose-derived MSCs (AD-MSCs) are more proliferative than BM-MSCs but have inferior osteogenic potential. 21Moreover, there are differences in the extracellular vesicles secreted by both the MSCs, with those derived from AD-MSCs having a higher influence on angiogenesis, whereas those derived from BM-MSCs contributing to the promotion proliferation. 45veral characteristics of pancreatic/islet-derived MSCs have also been reported.The pattern of chemokines and pro-regenerative factors in islet-derived MSCs is similar to that of BM-MSCs, and they have been shown to have immunomodulatory properties such as that of suppressing T cell proliferation in vitro. 37Islet-derived MSCs do not express insulin; however, compared to BM-MSCs, their level of histone modification of the insulin gene is closer to that of islets. 46Furthermore, some reports have suggested that MSCs established from pancreatic tissue may contribute to islet regeneration by producing more secreted substances involved in vascular development, wound healing, and chemotaxis than BM-MSCs. 44,47ditionally, transplantation of islet-derived MSCs into mice results in the differentiation of a number of cells into insulin-secreting cells. 32 this study, we found that npISLET-MSCs have a larger average diameter and proliferate less rapidly than npBM-MSCs.The characteristics of ISLET-MSCs, including secretory products and differentiation performance need to be clarified through further research.
It has been reported that MSC co-culture/co-transplantation may enhance the therapeutic effect of cells of interest, such as cells of the liver 9 and myocardium. 48Similarly, in pancreatic islets, the inhibition of apoptosis and enhancement of insulin secretory capacity of cultured Representative images (A).Colony-forming efficiency of cells (%) in CFU assay (B).There were no significant differences between the two groups.
islets via MSCs-derived exosomes has been reported when co-cultured with Wharton's jelly-derived MSCs in the umbilical cord. 49Furthermore, co-transplantation of BM-MSCs and islets has been reported to significantly reduce the need for postoperative insulin therapy. 10We believe that ISLET-MSCs are more effective than other MSCs for islet co-culture/transplantation based on the differentiation potential, the characteristics of cellular secretions, and the gene modification characteristics of ISLET-MSCs mentioned above.In the future, we intend to study islet co-cultivation/transplantation in conjunction with the characterization of the established npSILET-MSCs.
In this study, we developed a method of establishing MSCs from cryopreserved islets of clinical grade pigs.To pursue xenotransplantation therapy using pigs, it is necessary to suppress xeno-immune reactions in addition to managing donor pig hygiene.Thus, pig medical applications are being investigated, including the knockout of multiple antigen genes. 50Additionally, we have previously reported the establishment of MSCs from pigs lacking the α-Gal antigen. 51In the future, we plan to establish MSCs from pigs that are controlled and genetically regulated in a medical environment.This will improve donor supply and ensure stable cell quality for medical applications.

CONCLUSIONS
We have established a method for producing npISLET-MSCs from cryopreserved islets of clinical grade pigs.The preparation of npISLET-MSCs from cryopreserved islets enables a stable supply of cells of the same quality.Clinical grade neonatal porcine cryopreserved islets should be a promising source for future cell therapy using islet-derived MSCs.
pellet was gently resuspended in an MSC culture medium by pipetting up and down.The islets (2000 IEQ/well) were plated on a 6-well plate in 2 mL of MSC culture medium.The same MSC culture medium was used for both clinical and non-clinical grades of npISLET-MSCs: PRACTITIONER POINTS MSCs could be established from islets cryopreserved for more than 4 years.Characterization of MSCs established from cryopreserved islets of clinical grade neonatal pigs was conducted.Clinical grade neonatal porcine cryopreserved islets should be a promising source for future cell therapy using isletderived MSCs.
cium and magnesium ions (PBS (-)) and added 320 μL of 0.25% trypsin per well.The cells were kept in the incubator for 3 min at 37 • C.After the cells were detached, trypsin was neutralized with 1680 μL of MSC culture medium per well.The cell suspensions from four wells were F I G U R E 1 Micrographs of cultured cryopreserved clinical grade and non-clinical grade neonatal porcine islets (A).Islets cryopreserved for more than 4 years in Cellstor-S containing 10% DMSO were used.Islet (2000 IEQ/well) was cultured by plating 6-well plates with 2 mL MSC culture medium.collected into a 50 mL tube, and 32 mL (8 mL × 4 wells) of MSC culture medium was added.The cell suspensions were centrifuged for 5 min at 500 × g at approximately 22 • C. The cell pellet was resuspended in an MSC culture medium.The numbers of total and dead cells were counted.The cell suspensions (3 × 10 5 -6 × 10 5 total cells/flask) were replated into 75 cm 2 flasks in a 20 mL MSC culture medium.The 75 cm 2 flasks were placed in an incubator; the incubated cells were termed as passage 1 cells.

added 2 .
4 mL of 0.25% trypsin per 75 cm 2 flask.The cells were kept in the incubator for 3 to 5 min at 37 • C. The trypsin was neutralized with 12.6 mL of MSC culture medium per 75 cm 2 flask after the cells were detached.The cell suspensions were collected in a 50 mL tube.The cell suspensions were centrifuged for 5 min at 500 × g at approximately 22 • C .The isolated npISLET-MSCs were resuspended at 3 × 10 6 -6 × 10 6 cells/mL with Cellstor-S containing 10% DMSO or 4% propylene glycol.Approximately 1 mL of isolated npISLET-MSCs were placed in each cryovial.The cryovials were placed in a BICELL frozen at −80 • C • C water bath.To the thawed cell suspension, 10 mL of MSC culture medium was gently added.Furthermore, it was centrifuged for 5 min at 500 × g at approximately 22 • C .The supernatant was removed, and 10 mL of MSC culture medium was gradually added to the pellet.The pellet was gently resuspended in an MSC culture medium by pipetting up and down.We counted the number of total and dead cells.The cell suspensions (3 × 10 5 total cells/flask) were seeded into 75 cm 2 flasks in a 20 mL MSC culture medium.The 75 cm 2 flask was placed in the CO 2 incubator and incubated at 37 • C, 5% CO 2, and 90% humidity.Both clinical grade npISLET-MSCs and non-clinical grade npISLET-MSCs were approximately 90% confluent after 4 days of culture.When the cells reached 90% confluence, they were collected and replated into 75 cm 2 flasks or used for each experiment.We washed the cells with 8 mL PBS (-) and added 2.4 mL of 0.25% trypsin per 75 cm 2 flask.The cells were kept in the incubator for 3 to 5 min at 37 • C. Following the detachment of the cells, trypsin was neutralized with 12.6 mL of MSC culture medium per 75 cm 2 flask.The cell suspensions were collected in a 50 mL tube.The cell suspensions were centrifuged for 5 min at 500 × g at approximately 22 • C, and the supernatant was aspirated.The cells were suspended in an MSC culture medium and used for each experiment.
grade npISLET-MSCs and clinical grade npISLET-MSCs.The cell size of non-clinical grade npBM-MSCs was significantly smaller than that of non-clinical and clinical grade npISLET-MSCs (p <0.001).

F I G U R E 4
Mean diameters of non-clinical grade npBM-MSCs, non-clinical grade npISLET-MSCs, and clinical grade npISLET-MSCs.MSCs from passages 2 to 3 were used.The results of MSCs are shown as mean ± SD (n = 4).The diameter of non-clinical-grade npBM-MSCs is significantly smaller than that of non-clinical grade npISLET-MSCs and clinical grade npISLET-MSCs.Tukey's test ***p < 0.001.

F I G U R E 6
Differentiation potential of clinical grade npISLET-MSCs.Induction of adipogenic differentiation was evaluated using Oil Red staining (A).Induction of osteogenic differentiation was evaluated using alkaline phosphatase staining (B).

F I G U R E 7
Comparison of non-clinical grade npBM-MSCs and clinical grade npISLET-MSCs using Colony Forming Unit (CFU) assay.
4 mL ice-cold methanol for 15 min at 4 • C. To visualize colonies, cells were stained with 4 mL Giemsa diluted 1:19 with phosphate buffer for 30 min at room temperature and washed twice with H 2 O.The number of colonies with more than 50 cells was counted.The colony-forming efficiency of cells was calculated by dividing the number of colonies per dish by the number of cells (315) seeded per dish.