Human Blood Vessel–Derived Endothelial Progenitors for Endothelialization of Small Diameter Vascular Prosthesis

Background

Coronary bypass graft failure as a result of acute thrombosis and intimal hyperplasia has been the major challenge in surgical procedures involving small-diameter vascular prosthesis. Coating synthetic grafts with patients'' own endothelial cells has been suggested to improve the patency rate and overall success of bypass surgeries.

Methodology/Principal Findings

We isolated endothelial progenitor cells (EPCs) from leftover pieces of human saphenous vein/mammary artery. We demonstrate that EPCs can be expanded to generate millions of cells under low-density culture conditions. Exposure to high-density conditions induces differentiation to endothelial cell phenotype. EPC–derived endothelial cells show expression of CD144^high, CD31, and vWF. We then assessed the ability of differentiated endothelial cells to adhere and grow on small diameter expanded polytetrafluoroethylene (ePTFE) tubings. Since ePTFE tubings are highly hydrophobic, we optimized protocols to introduce hydrophilic groups on luminal surface of ePTFE tubings. We demonstrate here a stepwise protocol that involves introduction of hydrophilic moieties and coating with defined ECM components that support adhesion of endothelial cells, but not of blood platelets.

Conclusion/Significance

Our data confirms that endothelial progenitors obtained from adult human blood vessels can be expanded in vitro under xenoprotein-free conditions, for potential use in endothelialization of small diameter ePTFE grafts. These endothelialized grafts may represent a promising treatment strategy for improving the clinical outcome of small-caliber vascular grafts in cardiac bypass surgeries.     

Poly(styrenesulfonate)/poly(allylamine) multilayers: a route to favor endothelial cell growth on expanded poly(tetrafluoroethylene) vascular grafts

Small-diameter synthetic vascular grafts of expanded poly(tetrafluoroethylene) (ePTFE) polymer concern one of the most common alternatives for the replacement of diseased vessels. However, high failure rates arise especially due to the lack of endothelial cells (ECs). EC seeding was developed to build a monolayer on the luminal surface. Because ECs show little or no adhesion on synthetic prostheses, it is necessary to promote their retention. On ePTFE surfaces we successfully deposited polyelectrolyte multilayer films (PMFs) consisting of poly(ethylenimine) (PEI), poly(sodium 4-styrenesulfonate) (PSS), and poly(allylamine hydrochloride) (PAH) to obtain PEI-(PSS-PAH)3 films. EC adhesion and spreading on modified ePTFE were assessed by scanning electron and confocal microscopies. Cell viability was evaluated by Alamar Blue assay. After 7 days of culture, the ePTFE modified with PMF exhibited improvements of EC viability as compared to that of the controls (nonmodified ePTFE) or even ePTFE coated by a PAH monolayer (p < 0.05). Moreover, the spreading of ECs was largely enhanced compared to that of the same controls, resulting in a healthy confluent cell monolayer formation. Positive staining for the von Willebrand factor confirmed the EC phenotype. Promoting EC attachment and function on ePTFE modified with PMFs could become in the future a promising treatment for synthetic small-diameter vascular grafts.     

The long-term stability in gene expression of human endothelial cells permits the production of large numbers of cells suitable for use in regenerative medicine

Tissue engineering has been conducted in the study of cardiovascular grafts for many years. Many obstacles have been overcome in this rapidly changing field, but one difficulty has remained until now: the large number of endothelial cells (ECs) needed for seeding the inner layer of bypass graft. Recent advances in endothelial progenitor cell (EPC) isolation and culture techniques have increased the interest in genetic studies. Despite these advances in EPC studies, the "gold standard" for the seeding of tissue engineering constructs or hybrid grafts remains mature human umbilical vein endothelial cells (HUVECs). This study investigates the ability of HUVECs to be expanded in culture to provide sufficient cells for graft seeding. The levels of gene expression of key genes are then examined to ensure that these cells retain the EC phenotype. This study demonstrates that HUVECs may be cultured for up to 12 passages without alteration in phenotype. Subsequent passage numbers are sufficiently similar to those preceding them to allow cells of different passages to be mixed without gene expression anomalies.     

Enhancement of cell attachment and tissue integration by a IKVAV containing multi-domain peptide

Laminin contains a number of cell binding motifs including IKVAV and some that bind heparin. We developed a multi-domain synthetic peptide, LA2, which combines IKVAV sequences with a heparin-binding domain with the goal of improving cell attachment to otherwise non-adherent substrates. LA2 was used to coat polystyrene, ethyl vinyl acetate (EVA), expanded polytetrafluoroethylene (ePTFE), polycarbonate, titanium and stainless steel. In cell attachment studies, LA2 dramatically increased cell attachment to polystyrene and EVA compared to uncoated counterparts or those coated with SIKVAV. Similar increases were observed on ePTFE and titanium. On polystyrene, LA2 enhanced the attachment of endothelial cells, smooth muscle cells, epithelial cells, myoblasts, and osteoblast progenitor cells. Following adhesion, the cells underwent proliferation to form confluent monolayers with phenotypic morphologies. Using osteoblast progenitor cells (MC3T3 cells) grown on LA2/polystyrene, the cells exhibited an increased production of a differentiation marker, alkaline phosphatase. In vivo, LA2 improved tissue integration into ePTFE when implanted subcutaneously in rats. After 2 weeks, cells had penetrated deep into the LA2 coated ePTFE implant whereas little cell penetration was found in uncoated grafts. The implant sites exhibited little inflammation or other untoward effects. The results indicated that the LA2 peptide improved cell adhesion and tissue integration and might be useful in a number of tissue engineering applications.     

The endothelization of polyhedral oligomeric silsesquioxane nanocomposites

It has been recognized that seeding vascular bypass grafts with endothelial cells is the ideal method of improving their long-term patency rates. The aim of this study was to assess the in vitro cytocompatibility of a novel silica nanocomposite, polyhedral oligomeric silsesquioxane-poly(carbonate-urea)urethane (POSS-PCU) and hence elicit its feasibility at the vascular interface for potential use in cardiovascular devices such as vascular grafts. Using primary human umbilical vein endothelial cells (HUVEC), cell viability and adhesion were studied using AlamarBlue assays, whereas cell proliferation on the polymer was assessed using the PicoGreen dye assay. Cellular confluence and morphology on the nanocomposite were analyzed using light and electron microscopy, respectively. Our results showed that there was no significant difference between cell viability in standard culture media and POSS-PCU. Endothelial cells were capable of adhering to the polymer within 30 min of contact (Student's t-test, p<0.05) with no difference between POSS-PCU and control cell culture plates. POSS-PCU was also capable of sustaining good cell proliferation for up to 14d even from low seeding densities (1.0×10³ cells/cm²) and reaching saturation by 21 d. Microscopic analysis showed evidence of optimal endothelial cell adsorption morphology with the absence of impaired motility and morphogenesis. In conclusion, these results support the application of POSS-PCU as a suitable biomaterial scaffold in bio-hybrid vascular prostheses and biomedical devices.     

Decline of surface MHC I by adenoviral gene transfer of anti-MHC I intrabodies in human endothelial cells-new perspectives for the generation of universal donor cells for tissue transplantation

BACKGROUND: The seeding of small-calibre vascular polytetrafluoroethylene (PTFE) grafts with endothelial cells provides an increase in biocompatibility of the graft surface. The harvest and ex vivo culture of autologous endothelial cells is highly delicate. Allogeneic human umbilical vein endothelial cells (HUVEC) could be a potential cell source-however, rejection might occur due to major histocompatibility complex (MHC) I mismatches. Lowering cell surface MHC I expression on endothelial cells by gene transfer of an anti-MHC I intrabody might reduce graft failure. The intrabody consists of a single-chain variable fragment (sFv) of an anti-MHC I antibody, carrying a terminal KDEL sequence to retain the molecule together with the MHC I inside the endoplasmic reticulum. METHODS: Adenoviral gene transfer was used to express the intrabody in HUVEC. The MHC I surface expression was measured 48 h after transduction by flow cytometry. Functional effects of the intrabody expression were analyzed in a calcein release cytotoxicity assay. RESULTS: A transduction efficiency of more than 95% with EGFP-adenovirus indicates a sufficient gene transfer into HUVEC. Intrabody-adenovirus-transduced HUVEC show a massive reduction in MHC I surface expression creating almost a complete 'knockout' phenotype. Stimulation with inflammatory cytokines could not overcome this effect. The cell lysis of anti-MHC I intrabody-expressing HUVEC in a cytotoxicity assay is reduced when compared with the level of the MHC mismatched control. CONCLUSIONS: Our data indicate that HUVEC with reduced levels of MHC I might be used as universal donor cells for the seeding of vascular grafts.     

Micropatterned polymer surfaces improve retention of endothelial cells exposed to flow-induced shear stress

The use of synthetic polymeric vascular grafts is limited by the thrombogenecity of most biomaterials. Efforts to reduce thrombogenicity by seeding grafts with endothelial cells, the natural non-thrombogenic lining of blood vessels, have been thwarted by flow-induced cell detachment. We hypothesized that by creating well-defined micro-textured patterns on a surface, fluid flow at the surface can be altered to create discrete regions of low shear stress. We further hypothesized that, due to reduced shear stress, these regions will serve as sanctuaries for endothelial cells and promote their retention. To test these hypotheses, well-defined micro-textured polyurethane (PU) surfaces consisting of arrays of parallel 95-micron wide and 32-micron deep channels were created using an etched silicon template and solvent casting techniques. Based on computational fluid dynamics, under identical bulk flow conditions, the average local shear stress in the channels (46 dyn/cm2) was 28% lower than unpatterned surfaces (60 dyn/cm2). When PU surfaces pre-seeded with endothelial cells (EC) were exposed to the same bulk flow rate, EC retention was significantly improved on the micropatterned surfaces relative to un-patterned surfaces (92% vs. 58% retention).     

Perfusion seeding of channeled elastomeric scaffolds with myocytes and endothelial cells for cardiac tissue engineering

The requirements for engineering clinically sized cardiac constructs include medium perfusion (to maintain cell viability throughout the construct volume) and the protection of cardiac myocytes from hydrodynamic shear. To reconcile these conflicting requirements, we proposed the use of porous elastomeric scaffolds with an array of channels providing conduits for medium perfusion, and sized to provide efficient transport of oxygen to the cells, by a combination of convective flow and molecular diffusion over short distances between the channels. In this study, we investigate the conditions for perfusion seeding of channeled constructs with myocytes and endothelial cells without the gel carrier we previously used to lock the cells within the scaffold pores. We first established the flow parameters for perfusion seeding of porous elastomer scaffolds using the C2C12 myoblast line, and determined that a linear perfusion velocity of 1.0 mm/s resulted in seeding efficiency of 87% ± 26% within 2 hours. When applied to seeding of channeled scaffolds with neonatal rat cardiac myocytes, these conditions also resulted in high efficiency (77.2% ± 23.7%) of cell seeding. Uniform spatial cell distributions were obtained when scaffolds were stacked on top of one another in perfusion cartridges, effectively closing off the channels during perfusion seeding. Perfusion seeding of single scaffolds resulted in preferential cell attachment at the channel surfaces, and was employed for seeding scaffolds with rat aortic endothelial cells. We thus propose that these techniques can be utilized to engineer thick and compact cardiac constructs with parallel channels lined with endothelial cells. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

In Vitro Model of Tumor Cell Extravasation

Tumor cells that disseminate from the primary tumor and survive the vascular system can eventually extravasate across the endothelium to metastasize at a secondary site. In this study, we developed a microfluidic system to mimic tumor cell extravasation where cancer cells can transmigrate across an endothelial monolayer into a hydrogel that models the extracellular space. The experimental protocol is optimized to ensure the formation of an intact endothelium prior to the introduction of tumor cells and also to observe tumor cell extravasation by having a suitable tumor seeding density. Extravasation is observed for 38.8% of the tumor cells in contact with the endothelium within 1 day after their introduction. Permeability of the EC monolayer as measured by the diffusion of fluorescently-labeled dextran across the monolayer increased 3.8 fold 24 hours after introducing tumor cells, suggesting that the presence of tumor cells increases endothelial permeability. The percent of tumor cells extravasated remained nearly constant from1 to 3 days after tumor seeding, indicating extravasation in our system generally occurs within the first 24 hours of tumor cell contact with the endothelium.     

Magnetic beads (Dynabead™) toxicity to endothelial cells at high bead concentration: Implication for tissue engineering of vascular prosthesis

Magnetic beads (Dynabeads) have been used for the purification of endothelial cells. One application for this procedure may be for single-stage seeding of bypass grafts. The number of endothelial cells (EC) isolated is crucial and therefore to increase the number of cells extracted, a higher number of Dynabeads per cell may need to be used. The effect of large numbers of CD31 Dynabeads on cell proliferation/metabolism is unknown. We undertook this study using CD31-coated Dynabeads and EC from human umbilical vein. EC were coated at concentrations of 4, 10, or 50 beads per cell. The cells were cultured for 6 days with control being normal EC. Cellular proliferation was assessed by trypsinization of cells and metabolism assessed with an Alamar blue viability assay. In a further experiment a compliant polyurethane graft was single-stage seeded with both coated Dynabeads and normal EC. The results showed that using a higher number of beads per cell resulted in a reduction in cell proliferation and a reduction in cell metabolism. The total number of Dynabeads-coated cells in culture compared to controls (%) by day 6 were 30.7 +/- 2.56, 41.3 +/- 9.8 and 59.2 +/- 7.3 for 50, 10, and 4 beads per cell, respectively. The corresponding results for Alamar blue were 43.7 +/- 1.2, 61.8 +/- 1.4, and 72.1 +/- 4.3. The seeded grafts showed reduced metabolism with the Dynabeads-coated EC. In conclusion, high numbers of beads per cell have a late detrimental effect on cell proliferation and metabolism. Therefore for single-stage seeding lower numbers of Dynabeads will need to be used with resultant reduction in the number of available EC.     

旋转加压种植法在新型生物人工复合血管体外内皮化过程中的实验研究

目的：探讨在新型生物人工复合血管内腔面联合种植平滑肌细胞和内皮细胞的方法,比较研究旋转加压种植与普通灌注种植两种方法的内皮化效果,方法：先制备新型生物人工复合血管及获取培养鉴定平滑肌细胞和内皮细胞,再和旋转加压种植与普通灌注种植两种方法将平滑肌细胞和内皮细胞培养种植于新型复合血管内腔面,以光镜及扫描电镜等观察评价内皮化的效果。结果：旋转加压种植2小时末的复合血管腔内有大量内皮细胞,旋转加压种植9天后已形成完整的内皮细胞单层;普通灌注种植的复合血管内腔有内皮细胞附着,分布不均匀,未形成完整的内皮细胞层,结论：以旋转加压种植法在新型复合血管内腔面联合种植平滑肌细胞和内皮细胞效果满意,基本实现内皮化,可以满足复合血管内皮化的要求。     

Differential toxicities of air (mO-LDL) or copper-oxidized LDLs (Cu-LDL) toward endothelial cells.

In vivo low density protein (LDL) oxidation is a progressive phenomenon leading to the presence of minimally and highly oxidized LDLs in the subendothelial arterial space. Oxidized LDLs have been reported to be cytotoxic against endothelial cells. The goal of this study was to determine which of the minimally and highly oxidized LDLs were the most cytotoxic against bovine aortic endothelial cells (BAEC). Both the morphological aspect of the cells themselves, and LDH or MTT tests revealed that mO- or Cu-LDLs had similar cytotoxicity with up to 8 hours of oxidation, showing no relation with the level of LDL oxidation; for longer oxidation times, Cu-LDL cytotoxicity decreased. This phenomenon is linked to their different oxidation kinetics. Moreover, in the initial hours following BAEC incubation with mO- or Cu-LDLs, total cell glutathione dropped, whereas after 16 hours of incubation, highly oxidized Cu-LDL increased the glutathione level in the cell. The biphasic evolution of glutathione concentration corresponds to an autoprotective mechanism of cells against oxidized LDL cytotoxicity. This study suggests that the specific chemical characteristics of the different types of oxidized LDLs should always be precisely described in future assays devoted to studying the biological effects of what are known under the generic term as "oxidized LDLs". This precaution should prevent any confusion in interpreting different studies.     

In vivo biostability of four types of arterial grafts with impervious walls; their haemodynamic and pathological characteristics

We describe the haemodynamic and pathological characteristics of four types of impervious arterial prostheses, two alloplastic (Milrathane^® and Gore-Tex^®), and two chemically processed bovine heterografts (Solcograft^® and Solco P^®). They were implanted in the thoracic aortae of dogs for durations of 24 hours, 48 hours, one weeks, two weeks, one month, three months and six months. Haemodynamic analyses showed no relation between the shear rate index, I·_Y, and compliance, C_D. The observed shear rates are 6.5 times lower than those likely to damage the endothelial cell layer. Macroscopic and microscopic observations of explanted grafts showed the presence of obstructive thrombi at the anastomoses of Mitrathane^® grafts as early as one week. Gore-Tex^® grafts develop in the area of anastomoses parietal-thrombi which reorganize and become covered with pseudo-endothelial cells. The bovine heterografts show a similar behaviour. However, whereas Solcograft^® has an irregular thin wall, Solco P^® had improved characteristics except in the graft implanted for three months which demonstrated some manufacturing weaknesses. Both types showed the development of anastomotic pannus covered with endothelial-like cells. All grafts, whether alloplastic or chemically processed, suffered from an absence of healing of the middle part of the prosthesis. The cause of this problem will be found in the analysis of the biochemical and enzymatic reactations between the material used and its physiological surrounding.     

An in vitro study of cryopreserved and fresh human arteries: a comparison with ePTFE prostheses and human arteries studied non-invasively in vivo

The surgical options in arterial reconstruction are: the use of autologous arteries; autologous veins; or expanded polytetrafluoroethylene (ePTFE) grafts. However, the development of intimal hyperplasia when using veins or ePTFE grafts has been associated with graft failure. Since autologous arteries are not always available, the use of cryopreserved arteries has to be considered. The aims of this study were: (a) to compare the viscoelastic properties of stored cryopreserved arteries and fresh arteries by in vitro analysis; and (b) to compare the viscoelastic properties of arteries measured non-invasively in normotensive patients, with fresh arteries, cryopreserved arteries, and ePTFE segments. The viscoelastic studies were performed in normotensive patients using stress-strain analysis with non-invasive measurement of pressure and diameter in the common carotid artery, and in vitro measurements of pressure and diameter in arteries and prostheses. The in vitro studies showed that the elastic modulus (E), viscous modulus (eta), Stiffness Index (SI), Peterson modulus (Ep), and the pulse wave velocity (PWV) values for human cryopreserved carotid arteries were similar to the values obtained non-invasively in normotensive subjects (P>0.05) and to human fresh vessels (P>0.05). In vitro, the SI, Ep, PWV, and E values of ePTFE were significantly higher than the observed values in subjects and with fresh and cryopreserved arteries (P<0.05); on the other hand the ePTFE eta values were the lowest (P<0.05). We concluded that cryopreserved arteries have similar viscoelastic properties to those obtained in vivo in the arteries of normotensive subjects and in vitro in fresh arteries. Consequently, we conclude that the cryopreservation procedure does not modify the mechanical properties of the arterial wall.     

A microcarrier-based cocultivation system for the investigation of factors and cells involved in angiogenesis in three-dimensional fibrin matrices in vitro

Angiogenesis in situ includes coordinated interactions of various microvascular cell types, i.e., endothelial cells, pericytes and perivascular fibroblasts. To study the cellular interactions of microvascular cells in vitro, we have developed a microcarrier-based cocultivation system. The technical details of this method include seeding of endothelial cells on unstained cytodex-3 microcarriers and seeding of pericytes, fibroblasts or vascular smooth muscle cells on microcarriers which have been labeled by trypan blue staining. A mixture of both unstained and trypan blue-stained microcarriers was subsequently embedded in a three-dimensional fibrin clot. The growth characteristics of each cell type could be conveniently observed since the majority of cells left their supporting microcarriers in a horizontal direction to migrate into the transparent fibrin matrix. As differently stained microcarriers were randomly arranged in the fibrin matrix, the characteristic patterns of the microcarriers allowed location of particular points of interest at different developmental stages, facilitating the observation of cellular growth over the course of time. One further advantage of this microcarrier-based system is the possibility of reliably quantifying capillary growth by determination of average numbers of capillary-like formations per microcarrier. Thus, this model allows convenient evaluation of the effects of non-endothelial cells on angiogenesis in vitro. By using this coculture system, we demonstrate that endothelial capillary-like structures in vitro do not become stabilized by contacting vascular smooth muscle cells or pericytes during the initial stages of capillary formation.     

The effect of a silk Fibroin/Polyurethane blend patch on rat Vessels

Patch grafts are widely used in various kind of vascular surgeries such as detect repair or dilation of vascular stenosis. Expanded polytetrafluoroethylene (ePTFE) patches are flexible and handle well, but have shown problems with calcification as they are non-bioabsorbable and therefore permanently remain in the body. It is important to develop an alternative biocompatible patch. Silk fibroin (SF) was developed as a biocompatible material, but it lacks of the elasticity required for surgery as a patch. Polyurethane (PU) is also a well-known elastomer so this study focused on the SF and the PU blend materials with a weight ratio of 5:5 (SF/PU). To evaluate the SF/PU patch, the patches were implanted into the abdominal aortas of rats, using the ePTFE patch in the control group. Because it was more flexible the SF/PU patch was easier to implant than the ePTFE patch. At 1 week after implantation, the SF/PU patch had been infiltrated with cells and collagen fiber. The ePTFE control patch did not accumulate collagen fiber until 3 months and calcification occurred at 4 weeks. The SF/PU patch did not present any signs of calcification for 3 months. This study addressed the problems associated with using SF in isolation and showed that the SF/PU patch can be considered as a useful alternative to the ePTFE to overcome the problem of calcification.     

Multilayer rat hepatocyte aggregates formed on expanded polytetrafluoroethylene surface

Feasibility of using a macroporous membrane material, expanded polytetrafluoroethylene (ePTFE), for culturing hepatocytes on its surface was examined. Adult rat hepatocytes were attached to an ePTFE surface and cultured in a hormonally defined medium supplemented with or without fetal calf serum (FCS, 10%) or bovine serum albumin (BSA, 0.03–3%). When cultured in a FCS-suplemented medium, hepatocytes reorganized themselves into multilayer cell aggregates on an ePTFE surface. The morphological characteristics of hepatocytes were influenced by the modification of the ePTFE surface as well as the culture medium. Hepatocytes cultured on a polyvinylalcohol (PVA)-coated ePTFE surface formed many more multilayer cell aggregates than those cultured on an uncoated ePTFE surface. Such highly multilayered hepatocyte aggregates were also noted when the cells were cultivated in a BSA-supplemented medium. On the other hand, when cultured in a FCS- or BSA-free medium, hepatocytes formed cell monolayers on both PVA-coated and uncoated ePTFE surfaces as did the cells on a collagen-coated polystyrene surface. The hepatocytes in the aggregates exhibited high albumin expression capability and low DNA synthesis rate as compared with those in monolayer cultures. The multilayer hepatocyte aggregates, as immobilized on a PVA-coated ePTFE surface in a serum-supplemented medium, are shown to be not only morphologically, but functionally differentiated, and will provide us a model system for the development of a bioreactor using hepatocytes, particularly for a hybrid-type artificial liver. This revised version was published online in June 2006 with corrections to the Cover Date.     

Invasion of mesenchyme into three-dimensional collagen gels: a regional and temporal analysis of interaction in embryonic heart tissue

In normal heart development the endothelium of the atrioventricular canal, but not the ventricle, produces mesenchymal cells which seed (invade) into the intervening extracellular matrix toward the myocardium at around 64-69 hr of development. We have utilized three-dimensional collagen substrates to examine the initiation of seeding by atrioventricular canal endothelia in vitro and to compare and contrast the responses of the ventricular endothelia. Explants of atrioventricular canals and ventricles from staged embryos were placed on the surfaces of collagen gels prior to the onset of seeding in situ. At varied intervals of incubation, the explant was removed, leaving behind a monolayer on the surface of the gel which consisted of endothelial cells. Subsequently, the endothelial outgrowths were examined for seeded cells. The results confirm the regional endothelial differences seen in vivo. They also show that invasion of the collagen gels is due to an alteration in phenotype mediated by interaction with other components of embryonic heart explant. Lastly, the time course of this tissue interaction in vitro mimics the onset of seeding in vivo.     

Analysis of correlation between some parameters depending on cell proliferation and life span of "stationary phase aging" cultures and maximum life span of mammals

Earlier we developed a "stationary phase aging" model and introduced a definition of life span of "stationary phase aging" cell cultures. In this model the cells grow after seeding in flasks without subcultivation and medium change. They reach cell saturation density, stop dividing, gradually degrade ("stationary phase aging") and perish. By the term "culture life span" we designate the time from cell seeding until culture death. We designate the culture as dead when the number of living cells is less than 10 per cent of their number at saturation density of cell culture. The life span of transformed Chinese hamster cells was found to be proportional to the duration of their growth from cell seeding up to saturation density, as well as to the number of cell culture doublings and to be inversely proportional to the velocity of cell culture doubling for the same growth period. Maximum life span of mammals is known to be proportional to pregnancy duration and to the age at puberty. We found that maximal life span of mammals was proportional to the number of cell population doublings and inversely proportional to the velocity of cell population doubling during embryonal period or for the time from zygote to growth termination. The dependences for cell cultures and for mammals are analogous to each other.