Characterization of the cell growth analysis for detection of immortal cellular impurities in human mesenchymal stem cells.

The analysis of in vitro cell senescence/growth after serial passaging can be one of ways to show the absence of immortalized cells, which are frequently tumorigenic, in human cell-processed therapeutic products (hCTPs). However, the performance of the cell growth analysis for detection of the immortalized cellular impurities has never been evaluated. In the present study, we examined the growth rates of human mesenchymal stem cells (hMSCs, passage 5 (P = 5)) contaminated with various doses of HeLa cells, and compared with that of hMSCs alone. The growth rates of the contaminated hMSCs were comparable to that of hMSCs alone at P = 5, but significantly increased at P = 6 (0.1% and 0.01% HeLa) or P = 7 (0.001% HeLa) within 30 days. These findings suggest that the cell growth analysis is a simple and sensitive method to detect immortalized cellular impurities in hCTPs derived from human somatic cells.

ROR2 expression predicts human induced pluripotent stem cell differentiation into neural stem/progenitor cells and GABAergic neurons.

Despite the development of various in vitro differentiation protocols for the efficient derivation of specific cell types, human induced pluripotent stem cell (hiPSC) lines have varing ability to differentiate into specific lineages. Therefore, surrogate markers for accurately predicting the differentiation propensity of hiPSC lines may facilitate cell-based therapeutic product development and manufacture. We attempted to identify marker genes that could predict the differentiation propensity of hiPSCs into neural stem/progenitor cells (NS/PCs). Using Spearman's rank correlation coefficients, we investigated genes in the undifferentiated state, the expression levels of which were significantly correlated with the neuronal differentiation propensity of several hiPSC lines. Among genes significantly correlated with NS/PC differentiation (P < 0.01), we identified ROR2 as a novel predictive marker. ROR2 expression in hiPSCs was negatively correlated with NS/PC differentiation tendency, regardless of the differentiation method, whereas its knockdown enhanced differentiation. ROR2 regulates NS/PC differentiation, suggesting that ROR2 is functionally essential for NS/PC differentiation. Selecting cell lines with relatively low ROR2 expression facilitated identification of hiPSCs that can differentiate into NS/PCs. Cells with ROR2 knockdown showed increased efficiency of differentiation into forebrain GABAergic neurons compared to controls. These findings suggest that ROR2 is a surrogate marker for selecting hiPSC lines appropriate for NS/PC and GABAergic neuronal differentiations.

Stilbene analogs of resveratrol improve insulin resistance through activation of AMPK.

Resveratrol (RSV), 3,5,4'-trihydroxy-trans-stilbene, is known to have many beneficial physiological activities. We have synthesized several stilbene analogues and have reported that the hydroxyl group in the 4' position of RSV exhibited strong radical scavenging action. Using stilbene analogs, we investigated the structure of RSV to explain its protective effect against obesity and type 2 diabetes. All six analogs used in this study inhibited the differentiation of 3T3-L1 adipocytes. 3-Hydroxy-trans stilbene (3(OH)ST), and 3,4'-dihydroxy-trans stilbene (3,4'(OH)2ST) increased glucose uptake and induced adenosine monophosphate kinase (AMPK) phosphorylation in C2C12 myotubes independently of insulin. An in vivo study using mice fed high-fat diets indicated that 3(OH)ST was more effective than RSV in improving insulin resistance. In conclusion, RSV and its derivatives, particularly 3(OH)ST, inhibited adipocyte differentiation and enhanced glucose uptake in the myotubes, resulting in a reduction of obesity and an improvement in glucose tolerance in vivo.

[Regulatory science research to facilitate the development of cell/tissue-processed products].

Regenerative medicine is regarded as innovative therapy for severe diseases and damages caused by tissue loss and functional impairment. In Japan, regenerative medicine is one of the most important subjects issued by Council for Science and Technology Policy and also referred to in Medical Innovation of New Growth Strategy. Cell/tissue-processed products are living cells, which have been manipulated or processed for the purpose of regenerative medicine, and are extensively developing. Human somatic cells, somatic stem cells, embryonic stem cells, and induced pluripotent stem cells are cell sources used for regenerative medicine. Since we lack in experiences with cell/tissue-processed products, technical development of safety and quality assessment is urgently needed. National Institute of Health Sciences has carried out a mission of Regulatory Science and worked on safety assessment of pharmaceuticals and medical devices and their guideline development. The objective of our study is to develop safety and quality assessment methods for cell/tissue-processed products derived from stem cells, based on recent progresses in life science. We are currently developing methods to evaluate products as follows; a) useful and quantitative tumorigenicity tests to detect contamination of undifferentiated and/or abnormal cells in products, b) quality assessment by gene expression analysis and detection of genetic stability in a manufacturing process, and c) analysis of quality attributes associated with propensity of undifferentiated cells to set acceptable criteria of cell banks. We will be able to provide indicators to control the quality, efficacy and safety of stem cell-processed products and support efficient and economical promotion of the products. Especially, this study would help translate stem cell science into therapeutic products to patients with severe and life-threatening diseases, consequently contributing to administrative policy of Ministry of Health, Labor and Welfare.

Single-Cell RNA-Seq Reveals LRRC75A-Expressing Cell Population Involved in VEGF Secretion of Multipotent Mesenchymal Stromal/Stem Cells Under Ischemia.

Human multipotent mesenchymal stromal/stem cells (MSCs) have been utilized in cell therapy for various diseases and their clinical applications are expected to increase in the future. However, the variation in MSC-based product quality due to the MSC heterogeneity has resulted in significant constraints in the clinical utility of MSCs. Therefore, we hypothesized that it might be important to identify and ensure/enrich suitable cell subpopulations for therapies using MSC-based products. In this study, we aimed to identify functional cell subpopulations to predict the efficacy of angiogenic therapy using bone marrow-derived MSCs (BM-MSCs). To assess its angiogenic potency, we observed various levels of vascular endothelial growth factor (VEGF) secretion among 11 donor-derived BM-MSC lines under in vitro ischemic culture conditions. Next, by clarifying the heterogeneity of BM-MSCs using single-cell RNA-sequencing analysis, we identified a functional cell subpopulation that contributed to the overall VEGF production in BM-MSC lines under ischemic conditions. We also found that leucine-rich repeat-containing 75A (LRRC75A) was more highly expressed in this cell subpopulation than in the others. Importantly, knockdown of LRRC75A using small interfering RNA resulted in significant inhibition of VEGF secretion in ischemic BM-MSCs, indicating that LRRC75A regulates VEGF secretion under ischemic conditions. Therefore, LRRC75A may be a useful biomarker to identify cell subpopulations that contribute to the angiogenic effects of BM-MSCs. Our work provides evidence that a strategy based on single-cell transcriptome profiles is effective for identifying functional cell subpopulations in heterogeneous MSC-based products.

Evaluation of the reproducibility and positive controls of cellular immortality test for the detection of immortalized cellular impurities in human cell-processed therapeutic products.

Introduction: Contamination of human cell-processed therapeutic products (hCTPs) with tumorigenic/immortalized cellular impurities is a major concern in the manufacturing and quality control of hCTPs. The cellular immortality test based on cell growth analysis is a method for detecting tumorigenic/immortalized cellular impurities in hCTPs. However, the performance of the cellular immortality test has not yet been well characterized. In this study, we examined the reproducibility of the cellular immortality test in detecting HeLa cells as a model of tumorigenic cellular impurities, as well as the applicability of other models of cellular impurities with different tumorigenicity to the cellular immortality test. Methods: Using HeLa cells as a model for cellular impurities, we measured the growth rate of human mesenchymal stem cells (hMSCs) supplemented with HeLa cells at concentrations ranging from 0.01 to 0.0001% at each passage in three laboratories and evaluated the reproducibility of the detection of immortalized cellular impurities. In addition, HEK293 cells (another immortalized cell line) and MRC-5 cells (a non-immortalized cell line) were employed as cellular impurity models that exhibit different growth characteristics from HeLa cells, and the ability of the cellular immortality test to detect these different impurities when mixed with hMSCs was examined. Results: In the multisite study, the growth rate of hMSCs supplemented with 1 and 10 HeLa cells (0.0001% and 0.001%) significantly increased and reached a plateau in all three laboratories, whereas those of hMSCs alone eventually decreased. Moreover, when hMSCs were supplemented with 10 and 100 HEK293 and MRC-5 cells (0.001% and 0.01%), the growth rate significantly increased. The growth rate of hMSCs supplemented with HEK293 cells increased with passage and remained high, whereas that of hMSCs supplemented with MRC-5 cells eventually decreased, as in the case of hMSCs alone. Conclusions: These results indicate that the cellular immortality test is reproducible and can detect immortalized (i.e., potentially tumorigenic) cells such as HEK293 cells with a lower growth rate than HeLa cells by discriminating against normal cells, which could contribute to ensuring the safety and quality of hCTPs.

A selective cytotoxic adenovirus vector for concentration of pluripotent stem cells in human pluripotent stem cell-derived neural progenitor cells.

Highly sensitive detection of residual undifferentiated pluripotent stem cells is essential for the quality and safety of cell-processed therapeutic products derived from human induced pluripotent stem cells (hiPSCs). We previously reported the generation of an adenovirus (Ad) vector and adeno-associated virus vectors that possess a suicide gene, inducible Caspase 9 (iCasp9), which makes it possible to sensitively detect undifferentiated hiPSCs in cultures of hiPSC-derived cardiomyocytes. In this study, we investigated whether these vectors also allow for detection of undifferentiated hiPSCs in preparations of hiPSC-derived neural progenitor cells (hiPSC-NPCs), which have been expected to treat neurological disorders. To detect undifferentiated hiPSCs, the expression of pluripotent stem cell markers was determined by immunostaining and flow cytometry. Using immortalized NPCs as a model, the Ad vector was identified to be the most efficient among the vectors tested in detecting undifferentiated hiPSCs. Moreover, we found that the Ad vector killed most hiPSC-NPCs in an iCasp9-dependent manner, enabling flow cytometry to detect undifferentiated hiPSCs intermingled at a lower concentration (0.002%) than reported previously (0.1%). These data indicate that the Ad vector selectively eliminates hiPSC-NPCs, thus allowing for sensitive detection of hiPSCs. This cytotoxic viral vector could contribute to ensuring the quality and safety of hiPSCs-NPCs for therapeutic use.

Effects of intracerebral microinjection of hydroxylated-[60]fullerene on brain monoamine concentrations and locomotor behavior in rats.

Fullerenes are condensed ring aromatic compounds with extended pi systems; they have unique cage structures. Current studies suggest that several fullerene derivatives have neuroprotective effects, and it is expected that fullerenes will be useful in drug delivery system and novel medical devices targeting the brain. However, little is known about the effects of fullerenes and its derivative on brain function. We examined the effect of fullerene(OH)24 on the central nervous system in this study. In a V79 colony assay, the IC50 of fullerene(OH)24 was 1.74 microg/ml. In an MTT assay, fullerene(OH)24 reduced proliferation of normal human astrocytes obviously. In an vivo study, 0.25 mg/kg(-1) of fullerene(OH)24 was injected into the lateral ventricle of rat brains. The intracerebral injection of fullerene(OH)24 remarkably decreased body weight and locomotor behavior of rats on day 1, but drastically increased locomotor behavior on day 7. The intracerebral injection of fullerene(OH)24 changed the monoamine concentration greatly on day 1 and slightly on day 30 after the injection. These results suggest that intracerebral injection of fullerene(OH)24 had strong and acute effects on the central nervous system, but that the effects were not permanent. In conclusion, we suggest that fullerene's derivative, fullerene(OH)24 had toxic effects on brain cells and that intracerebral injection of fullerene(OH)24 had acute harmful effects on brain monoamines neurotransmission and locomotor activity.

[Microarray analysis of the effects of serum-free medium on gene expression changes in human mesenchymal stem cells during the in vitro culture].

We examined the effects of serum-free medium on the gene expression changes in human mesenchymal stem cells (hMSCs) during the in vitro culture using a DNA microarray analysis. In this study, we cultured hMSCs with two kinds of medium; 1) MSCGM (contain 10% fetal bovine serum) or 2) STK2 (serum-free medium developed for mesechymal stem cells multiplication), and compared hMSCs proliferation, cell morphology, and gene expression changes until 50 days culture. Expression analysis was performed with Affymetrix GeneChip Human Genome U133 Plus 2.0 Array. hMSC proliferation was significantly higher in STK2 medium than in MSCGM medium. The cell morphology of hMSC cultured with STK2 was not significantly changed in 50 days culture. The gene expression changes in hMSCs during the in vitro culture were significantly higher in STK2 than in MSCGM. After 50 days culture, 1991 genes were significantly changed the expression levels compared with 3 days in STK2 but not MSCGM. The expressions of genes related to cell cycle, cancer, proliferation, and cell growth were significantly changed by STK2 for 50 days culture. It was also changed by STK2 that the expressions of genes related to the signaling pathways contain various growth factors, such as IGF-1, FGF, TGF-ß, EGF, proliferation, and cell cycle. These results suggest that STK2 may be useful to obtain an enough number of hMSC cells for tissue engineered medical devices in short-term, however, it should be recognized that STK2 would alter the expressions of genes related to a variety of signaling pathways in hMSC if the culture period would be extended to obtain a large number of cells.

[Effectivity of the novel serum-free medium STK2 for proliferating human mesenchymal stem cells].

To apply human mesenchymal stem cells (hMSC) to regenerative medicines, it is necessary to multiply hMSC in vitro in a short period. In addition, it is desirable that the medium which is used for the hMSC multiplication is not supplemented with the serum, because the addition of the serum has risks of infection. In this study, we cultured hMSC with three kinds of medium used for multiplying hMSC (DMEM, MSCGM, STK2) and compared hMSC proliferation in each medium. As a result, it was confirmed that hMSC proliferation was significantly higher in STK2 medium which is a novel serum-free medium developed for hMSC multiplication. Moreover, we compared the hMSC proliferation in these media under the environment that assumed bone reproduction. When we cultured hMSC in each medium with hydroxyapatite (HAp), the proliferative inhibition by HAp depended on the additive amount, and the degree of the proliferative inhibition was different among the media but the lowest inhibitory effect was observed in STK2 medium.

Development of selective cytotoxic viral vectors for concentration of undifferentiated cells in cardiomyocytes derived from human induced pluripotent stem cells.

Cell-processed therapeutic products (CTPs) derived from human pluripotent stem cells (hPSCs) have innovative applications in regenerative medicine. However, undifferentiated hPSCs possess tumorigenic potential; thus, sensitive methods for the detection of residual undifferentiated hPSCs are essential for the clinical use of hPSC-derived CTPs. The detection limit of the methods currently available is 1/105 (0.001%, undifferentiated hPSCs/differentiated cells) or more, which could be insufficient for the detection of residual hPSCs when CTPs contain more than 1 × 105 cells. In this study, we developed a novel approach to overcome this challenge, using adenovirus and adeno-associated virus (AdV and AAV)-based selective cytotoxic vectors. We constructed AdV and AAV vectors that possess a suicide gene, iCaspase 9 (iCasp9), regulated by the CMV promoter, which is dormant in hPSCs, for the selective expression of iCasp9 in differentiated cells. As expected, AdV/CMV-iCasp9 and AAV/CMV-iCasp9 exhibited cytotoxicity in cardiomyocytes but not in human induced pluripotent stem cells (hiPSCs). The vectors also induced apoptosis in hiPSC-derived cardiomyocytes, and the surviving cells exhibited higher levels of hPSC marker expression. These results indicate that the AdV- and AAV-based cytotoxic vectors concentrate cells expressing the undifferentiated cell markers in hiPSC-derived products and are promising biological tools for verifying the quality of CTPs.

The effect of sulfated hyaluronan on the morphological transformation and activity of cultured human astrocytes.

We demonstrated the effect of synthesized sulfated hyaluronan (SHya), which is composed of a sulfated group and hyaluronan, and basic fibroblast growth factor 2 (FGF-2) on normal human astrocytes (NHA) activity and its morphological transformation in vitro study. Astrocyte is a kind of glial cell and stellated astrocyte (activating astrocyte) supports axons network, neurons survival and synaptic plasticity. Treatment of SHya hardly affected NHA proliferation. However combination treatment of SHya and FGF-2 increased NHA proliferation. Treatment of SHya promoted transformation of normal astrocyte into a stella morphology (stellation) and combination treatment of SHya and FGF-2 promoted stellation than that of SHya only. Treatment of SHya increased glial fibrillary acidic protein (GFAP), nestin mRNA and GFAP protein expression in the stellated NHA. The cell-cell adhesion of NHA increased by treatment of SHya. Treatment of SHya increased heparin-binding trophic factors FGF-2, midkine, and some other trophic factors mRNA level in the NHA. These results suggested that the treatment of SHya promoted NHA activity due to enhancing neurotrophins production and the morphological transformation of NHA and the effect of SHya on astrocytes partly involved FGF-2 activity. These findings indicate that SHya may be involved in the astrocyte activity and support neurons survivals.

The metabolism and distribution of docosapentaenoic acid (n-6) in the liver and testis of growing rats.

To investigate the metabolism and distribution of docosapentaenoic acid (22:5n-6, DPA) in the liver and testis of growing rats, 22:5n-6 was administered to their dams. Newborn rats with a low hepatic arachidonic acid (20:4n-6, AA) level were generated by administrating a diet rich in docosahexaenoic acid (22:6n-3, DHA) but n-6 fatty acid (FA) free to pregnant dams. After parturition, 22:5n-6 or linoleic acid (18:2n-6, LA) was administered with a high level of 22:6n-3 to the dams until weaning. At weaning, the hepatic 20:4n-6 level was significantly highest in the DPA-DHA but not LA-DHA diet-fed animals. The hepatic delta-6 desaturase (D6D) mRNA abundance was significantly lower in both the LA-DHA and DPA-DHA diet-fed animals, connoted with the 20:4n-6 content recovered by 22:5n-6 that did not involve D6D and supporting the occurrence of retroconversion in the liver of the growing rats. The low D6D level in the 3-week-old testis was not in proportion to the elevated 22:5n-6 level, implying that early testicular 22:5n-6 accumulation might require supply from the circulation system.

Effects intracerebral microinjection and intraperitoneal injection of [60]fullerene on brain functions differ in rats.

Fullerenes are condensed ring aromatic compounds with extended pi systems and unique cage structures. Fullerenes are used for medical devices such as carbon nanotubes because they are very flexible and suitable for drug delivery systems. Recently, fullerene derivatives and tube-shaped materials have been used for neuroregeneration studies, and we expect that fullerenes and carbon nanotubes have potential uses as materials in novel medical devices targeting the brain. However, little information on the effects of fullerenes on brain function is available; thus, we examined the effects of [60]fullerene (C60) on the central nervous system in this study. In a V79 cell colony Asia, the IC50 of C60 was 1620 microg/ml. In an in vivo study, 0.25 mg/kg B.W. of C60 was injected into the lateral brain ventricle or abdominal cavity of rats. The intracerebral injection of C60 increased the locomotor behavior of the rats on days 1 and 30 after the injection. The intraperitoneal injection of C60 did not change the locomotor behavior of rats acutely, but it was decreased on day 30. The intracerebral injection of C60 affected monoamine concentrations in the rat brain. In particular, serotonin turnover rates were increased in the hypothalamus, cerebral cortex, striatum, and hippocampus, and dopamine turnover rates were increased in the hypothalamus, cerebral cortex, and striatum. The intraperitoneal injection of C60 decreased only the dopamine turnover rate in the hippocampus. These results suggest that intracerebral injection of C60 had different effects on the central nervous system than intraperitoneal injection. In conclusion, it was suggested that fullerene did not cross the blood-brain barrier. The intracerebral injection of C60 affected neurotransmission in the brain widely, and the monoamine dysbolism might be related to changes in locomotor activity.

[Change in characteristics of human mesenchymal stem cells during the in vitro culture -- c-myc gene expression and chromosome aberrations at the c-myc locus].

We investigated mRNA expression of c-myc and chromosome aberrations at the c-myc locus in the same passage number of human mesenchymal stem cells (hMSCs). To understand the sensitivity of mRNA expression and the induction of chromosome aberrations, we first tested them in hMSC and cancer cell lines (HeLa S3, HOS, and OUMS-27). The c-myc mRNA expressions in HeLa S3 and OUMS-27 were significantly higher than those in hMSC, but then those in HOS were not. On the other hand, c-myc aberrant cells detected by fluorescence in situ hybridization in HeLa S3, HOS, and OUMS-27 were significantly higher than that in hMSC. Both analyses were performed in hMSCs derived from five donors for the culture period of 50 days. In hMSCs from one donor, the frequency of c-myc aberrant cells significantly increased at 20 and 50 days respectively, and each mRNA expressions had a tendency to increase, but there is no significant change among 3, 20 and 50 days. In hMSCs from the others, both endpoints did not change for 50 days. For safe use of somatic stem cells in the regenerative medicine, the investigation of characteristic change of them during the in vitro culture is important. In the present study, we showed the mRNA expressions and chromosome aberrations of hMSCs in in vitro culture as the first step for establishing of safety evaluation of tissue engineered medical devices using normal hMSCs.

[Safety evaluation of tissue engineered medical devices using normal human mesenchymal stem cells].

Several recent studies demonstrated the potential of bioengineering using somatic stem cells in regenerative medicine. Adult human mesenchymal stem cells (hMSCs) derived from bone marrow have the pluripotency to differentiate into cells of mesodermal origin, e.g., bone, cartilage, adipose, and muscle cells; they, therefore, have many potential clinical applications. On the other hand, stem cells possess a self-renewal capability similar to cancer cells. For safety evaluation of tissue engineered medical devices using normal hMSCs, in this study, we investigated the expression levels of several genes that affect cell proliferation in hMSCs during in vitro culture. We focused on the relationship between the hMSC proliferation and their transforming growth factor beta (TGFbeta) signaling during in vitro culture. The proliferation rate of hMSCs gradually decreased and cellular senescence was observed for about 3 months. The mRNA expressions of TGFbeta1, TGFbeta2, and TGFbeta receptor type I (TGFbetaRI) in hMSCs increased with the length of cell culture. The mRNA expressions of Smad3 increased, but those of c-myc and nucleostemin decreased with the length hMSCs were in in vitro culture. In addition, the expression profiles of the genes which regulate cellular proliferation in hMSCs were significantly different from those of cancer cells. In conclusion, hMSCs derived from bone marrow seldom underwent spontaneous transformation during 1-2 months in vitro culture for use in clinical applications. In hMSCs as well as in epithelial cells, growth might be controlled by the TGFbeta family signaling.

Effects of a biodegradable polymer synthesized with inorganic tin on the chondrogenesis of human articular chondrocytes.

Recent study has shown that biodegradable polymers are attractive candidates for chondrocyte fixation and further transplantation in cartilage tissue engineering. Poly (glycolic acid) (PGA), a polymer of glycolic acid, is widely used in orthopedic applications as a biodegradable polymer. Organotin, lead, antimony, and zinc are catalysts commonly used in synthesizing PGA. Here, we investigated the biocompatibility of PGA, synthesized with and without inorganic tin as a catalyst in chondrogenesis of human articular chondrocytes in a micromass culture system. Significant enhancement of chondrocyte proliferation and expression of the collagen type II protein gene were observed in cultures treated with PGA synthesized with a tin catalyst. However, aggrecan gene expression was very similar to the control culture. Amount of collagen type II protein was also increased in the same group of cultured chondrocytes. In contrast, PGA without a catalyst caused overall inhibition of chondrogenesis. Despite several positive findings, extensive investigations are essential for the feasibility of this PGA(Sn) in future clinical practice.

Application of cell growth analysis to the quality assessment of human cell-processed therapeutic products as a testing method for immortalized cellular impurities.

In human cell-processed therapeutic products (hCTPs) for clinical application, tumorigenic cellular impurities in the manufacturing process are a major concern. Because cellular immortalization is one of the prerequisite steps in tumorigenesis, we tested whether cell growth analysis can be employed to check for immortalized (and potentially tumorigenic) cellular impurities in hCTPs. We monitored the growth of human bone marrow-derived mesenchymal stem cells (BMSCs) mixed with HeLa cells at a ratio of 1/106 or more and compared their growth rates with that of BMSCs alone. The cell growth analysis detected a significant increase in the growth rate of the BMSCs spiked with 0.0001% HeLa within 30 days at a probability of 47%. When human adipose-derived stem cells (ADSCs) were spiked with ASC52telo cells, a human telomerase reverse transcriptase (hTERT)-immortalized adipose-derived mesenchymal stem cell line, at a ratio of 0.001% or more, their growth rates were significantly increased within 15 passages, compared with that of ADSCs alone. These results indicate that cell growth analysis for the detection of immortalized cellular impurities in human somatic stem cells is simple and can be useful for the quality assessment of hCTPs in the manufacturing process.

In Vitro Endothelialization Test of Biomaterials Using Immortalized Endothelial Cells.

Functionalizing biomaterials with peptides or polymers that enhance recruitment of endothelial cells (ECs) can reduce blood coagulation and thrombosis. To assess endothelialization of materials in vitro, primary ECs are generally used, although the characteristics of these cells vary among the donors and change with time in culture. Recently, primary cell lines immortalized by transduction of simian vacuolating virus 40 large T antigen or human telomerase reverse transcriptase have been developed. To determine whether immortalized ECs can substitute for primary ECs in material testing, we investigated endothelialization on biocompatible polymers using three lots of primary human umbilical vein endothelial cells (HUVEC) and immortalized microvascular ECs, TIME-GFP. Attachment to and growth on polymer surfaces were comparable between cell types, but results were more consistent with TIME-GFP. Our findings indicate that TIME-GFP is more suitable for in vitro endothelialization testing of biomaterials.