Human macrovascular endothelial cells: Optimization of culture conditions

Summary The purpose of this study is to identify optimal culture conditions to support the proliferation of human macrovascular endothelial cells. Two cell lines were employed: human saphenous vein endothelial cells (HSVEC) and human umbilical vein endothelial cells (HUVEC). The influence of basal nutrient media (14 types), fetal bovine serum (FBS), and mitogens (three types) were investigated in relation to cell proliferation. Additionally, a variety of extracellular matrix (ECM) substrate-coated culture dishes were also tested. The most effective nutrient medium in augmenting cell proliferation was MCDB 131. Compared to the more commonly used M199 medium, MCDB 131 resulted in a 2.3-fold increase in cell proliferation. Media containing 20% FBS increased cell proliferation 7.5-fold compared to serum-free media. Among the mitogens tested, heparin (50 μg/ml) and endothelial cell growth supplement (ECGS) (50μg/ml) significantly improved cell proliferation. Epithelial growth factor (EGF) provided no improvement in cell proliferation. There were no statistical differences in cell proliferation or morphology when endothelial cells were grown on uncoated culture plates compared to plates coated with ECM proteins: fibronectin, laminin, gelatin, or collagen types I and IV. The culture environment yielding maximal HSVEC and HUVEC proliferation is MCDB 131 nutrient medium supplemented with 2 mM glutamine, 20% FBS, 50 μg/ml heparin, and 50 μg/ml ECGS. The ECM substrate-coated culture dishes offer no advantage.     

Heparin inhibits SMC growth in the presence of human and fetal bovine serum

Heparin (HP) has antiproliferative as well as anticoagulant properties, but not all HP preparations are equally antiproliferative. A recent report found that HP lost its total antiproliferative activity when fetal bovine serum (FBS) was replaced with human serum (HS) in culture media. This observation led to the investigation of our most potent antiproliferative Upjohn HP preparation effects on bovine pulmonary artery smooth muscle cells (PASMC) and systemic SMC growth stimulated in the presence of either FBS or HS. Bovine PASMC, human PASMC, and bovine aortic SMC were treated with 10 microg/ml Upjohn HP in either 15% FBS or 15% HS and the cell number was determined by a Coulter counter. We found that Upjohn HP significantly inhibited bovine PASMC and systemic SMC proliferation in both HS and FBS. The antiproliferative activity of the above HP preparation in HS may lead to an effective treatment of pulmonary vascular and systemic remodeling.     

Effects of fetal bovine serum, FSH and 17beta-estradiol on the culture of bovine preantral follicles

We describe a 7-d culture in droplets of collagen gel of isolated small bovine preantral follicles in medium with or without 10% fetal bovine serum (FBS). In addition, the effect of human recombinant FSH and 17beta-estradiol on the morphology and growth of the preantral follicles was investigated in medium without FBS. After culture in medium with 10% FBS, the increase in follicle diameter was 13.1 +/- 8.4 microm, the percentage of BrdU-labeled cells was 49.9 +/- 11.3 and the number of cells per area granulosa was 11.1 +/- 1.8. Omission of serum from the culture medium had no effect on the percentage of labeled cells, but the diameter increase was lower and the cells were smaller. Apparently, serum affects the size of the granulosa cells from small preantral follicles rather than the stimulation of cell proliferation. Addition of human recombinant FSH and/or 17beta-estradiol to serum-free medium resulted in a larger diameter increase during culture compared with that of the control. With FSH, this was due to an increase in cell proliferation, while with estradiol this was caused by an increase in granulosa cell size. The effects of simultaneous treatment with FSH and estradiol was simply the combination of their individual effects. In conclusion, small bovine preantral follicles can be cultured for 7 d in the absence of serum and hormones. The follicles increase in diameter and react to FSH with enhanced cell proliferation and to estradiol with an increase in cell size.     

人工基膜对鼻咽癌上皮细胞株（CNE—2）生长的影响

人工基膜（ＡＢＭ）主要以Ⅰ型胶原的水合性胶原丝网为网架,辅上纤维连结蛋白,Ⅳ型胶原和层粘连蛋白等主要基膜糖蛋白制备而成,具海绵状的形态结构。ＡＢＭ可减少胎牛血清用量１０％,提高细胞生活力和延长细胞传代周期。在２－５％血清浓度的情况下,ＡＢＭ可提高ＣＮＥ－２细胞的生长效率,克隆形成率和克隆生长率而抑制细胞的＾３Ｈ－ＴｄＲＡ掺入。提示在体外研究细胞外基质对细胞的影响时应使用低血清培养液。ＡＢＭ是体外诱     

Porcine platelet lysate as a supplement for animal cell culture

A novel supplementation of cell growth media based on a porcine platelet lysate was developed for culture of animal-derived cells. The platelet lysate was produced from porcine blood and contained lysate of platelets and plasma components. It showed satisfactory microbiological integrity and it carried only low amount of endotoxins (<10 EU/mL). The porcine platelet lysate supported well proliferation of Vero (African green monkey transformed kidney epithelial cells), Chinese hamster ovary (CHO) and hybridoma cells comparable to fetal bovine serum (FBS). Platelet lysate shows promise as a viable choice over FBS as it can be produced in large quantities, high lot-to-lot consistency and with an attractive price structure. Furthermore it is a strong alternative to FBS for ethical reasons. It is expected that it can be used as a general supplementation for most animal cells for research studies on the proliferation of cells and their expression of products.     

Serum-free culture conditions for the growth of normal rat mammary epithelial cells in primary culture

Summary A monolayer culture system has recently been developed for the extended growth and serial passage of normal rat mammary epithelial (RME) cells. In this system the cells undergo greater than 20 population doublings when grown on type I collagen-coated tissue culture dishes in Ham's F12 medium supplemented with insulin, hydrocortisone, epidermal growth factor, prolactin, progesterone, cholera toxin, and 5% fetal bovine serum (FBS). The purpose of the present studies was to define additional growth factors that would allow equivalent RME cell proliferation in serum-free medium. Ethanolamine (EA) was effective at reducing the FBS requirements for RME cell proliferation and at its optimum concentration did so by greater than 20-fold. Even with optimum levels of EA there was essentially no cell proliferation in the absence of FBS. However, addition of bovine serum albumin (BSA) to the hormone, growth factor, and EA-supplemented medium resulted in substantial proliferation in the absence of serum, and the further addition of transferrin (T) potentiated this effect. Thus, in this culture system, replacement of FBS with EA, BSA, and T resulted in RME cell proliferation in primary culture which was equivalent to that obtained in the 5% FBS-containing medium. This work was supported by grant RR-05529 from the Division of Research Resources, National Institutes of Health, Bethesda, MD, and by Public Health Service grant CA40064-01 from the National Cancer Institute, Bethesda, MD.     

Effect of indomethacin and lactoferrin on human tenocyte proliferation and collagen formation in vitro

Non-steroidal anti-inflammatory drugs (NSAIDs) are widely used in patients with injuries and inflammation of tendon and ligament, and as post-surgical analgesics. The aim of this study is to investigate the effect of indomethacin, a classic NSAID and its combinational effect with an anabolic agent of skeletal tissue, lactoferrin, on the proliferation and collagen formation of human tenocytes in vitro. A factorial experimental design was employed to study the dose-dependent effect of the combination of indomethacin and lactoferrin. The results showed that indomethacin at high concentration (100 μM) inhibited human tenocyte proliferation in culture medium with 1–10% fetal bovine serum (FBS) in vitro. Also, high dose of indomethacin inhibited the collagen formation of human tenocytes in 1% FBS culture medium. Lactoferrin at 50–100 μg/ml promoted human tenocyte survival in serum-free culture medium and enhanced proliferation and collagen synthesis of human tenocytes in 1% FBS culture medium. When 50–100 μg/ml lactoferrin was used in combination with 100–200 μM indomethacin, it partially rescued the inhibitory effects of indomethacin on human tenocyte proliferation, viability and collagen formation. To our knowledge, it is the first evidence that lactoferrin is anabolic to human tenocytes in vitro and reverses potential inhibitory effects of NSAIDs on human tenocytes.     

Optimization of the processing of porcine platelet-rich plasma and its application on human mesenchymal stem cell cultivation

Because of ethical and scientific controversy, the utilization of fetal bovine serum (FBS) for cell culture medium must be minimized. This study develops porcine platelet-rich plasma (P-PRP) as a FBS substitute for human mesenchymal stem cell (hMSC) cultivation. Concentrating porcine blood by serial centrifugation to obtain P-PRP leads to activation by different agonist combinations to stimulate the secretion of growth factors. The concentration of growth factor in P-PRP is significantly increased by activation (p < 0.05). The concentration of PDGF, KGF and TGF-β in activated P-PRP is significantly higher than that in FBS. Design-expert was used to decide whether Co−T+Ca−, Co+T−Ca−, and Co+T+Ca− are optimal agonist formulations. MSC cultivation shows that the attachment rate, proliferation rate and viability of P-PRP supplemented media are significantly higher than those for FBS-supplemented and commercial media (p < 0.05). The results demonstrate that P-PRP is an optimal FBS substitute that supports in vitro h-MSCs expansion for subsequent biomedical applications.     

Expansion of human bone marrow-derived mesenchymal stromal cells: serum-reduced medium is better than conventional medium

Background aimsHuman mesenchymal stromal cells (hMSC) are of enormous interest for various clinical applications. For the expansion of isolated hMSC to relevant numbers for clinical applications, 10% fetal bovine serum (FBS)-supplemented medium is commonly used. The main critical disadvantage of FBS is the possibility of transmission of infectious agents as well as the possibility of immune rejection of the transplanted cells in response to the bovine serum. Therefore, we tested a commercially available medium, Panserin 401, that was specifically developed for serum-free cell cultivation.MethodshMSC were isolated from bone marrow (BM) and expanded in either Dulbecco's modified Eagle medium (DMEM) or Panserin 401 alone, or combined with FBS (2% or 10%), with or without supplementary growth factors. Cell proliferation and cytotoxicity were monitored twice a week for 3 weeks.Results and ConclusionsNo proliferation was observed in any of the serum-free media. However, DMEM/10% FBS (the conventional culture medium for hMSC) and DMEM/2% FBS with growth factors revealed moderate proliferation. Interestingly, the best proliferation was obtained using Panserin 401 supplemented with 2% FBS and growth factors (as well as with 10% FBS). Analysis of cell growth in Panserin 401 supplemented with 2% FBS only or with growth factors only revealed no proliferation, demonstrating the necessity of the combination of 2% FBS and growth factors. Efficient isolation and expansion of hMSC from cancellous bone could also be performed using Panserin 401 with 2% FBS and growth factors. Furthermore, these isolated cultures maintained multipotency, as demonstrated by adipogenic and osteogenic differentiation.     

Effects of ascorbic acid and ascorbic acid 2-phosphate, a long-acting vitamin C derivative, on the proliferation and differentiation of human osteoblast-like cells

In order to investigate the effect of ascorbic acid (AsA) and ascorbic acid 2-phosphate (Asc 2-P), a long-acting vitamin C derivative, on the growth and differentiation of human osteoblast-like cells, we supplemented the culture medium of MG-63 cells with various concentrations (0.25 to 1 mM) of these factors. Asc 2-P significantly stimulated nascent cell growth at all concentrations in the presence of fetal bovine serum (FBS). On the other hand, AsA showed a growth repressive effect depending on its concentration, and that of FBS. Asc 2-P also increased expression of osteoblast differentiation markers, such as collagen synthesis and alkaline phosphatase (ALP) activity. These stimulative activities of Asc 2-P were attenuated by inhibitors of collagen synthesis, indicating that these effects were dependent on collagen synthesis. Electron micrographs of the cells showed the formation of a three-dimensional tissue-like structure endowed with a mature extracellular matrix in the presence of Asc 2-P.     

An antiproliferative heparan sulfate species produced by postconfluent smooth muscle cells

Heparan sulfate was isolated form the cell surface, cell pellet, and culture medium of exponentially growing as well as postconfluent bovine aortic smooth muscle cells (SMCs). After chromatography on DEAE-Sephadex and Sepharose 4B, the various mucopolysaccharides were examined for their ability to cause growth inhibition in a SMC bioassay. The heparan sulfate isolated from the surface of postconfluent SMCs possessed approximately eight times the antiproliferative potency per cell of the heparan sulfate obtained from the surface of exponentially growing SMCs. Heparan sulfate isolated from other fractions of exponentially growing or postconfluent SMCs possesses little growth inhibitory activity. The difference in the antiproliferative activities of heparan sulfate obtained from the surface of SMCs in the two growth states could not be attributed to the synthesis of a greater mass of mucopolysaccharide by postconfluent SMCs. Indeed, heparan sulfate isolated from the surface of the postconfluent SMCs exhibits a specific antiproliferative activity which is 13-fold greater than mucopolysaccharide obtained from the surface of exponentially growing SMCs and more than 40-fold greater than commercially available heparin. In addition, exponentially growing SMCs did not exhibit an enhanced ability to degrade the complex carbohydrate. Furthermore, other investigations indicate that the small amount of growth inhibitory activity intrinsic to heparan sulfate isolated from the surface of exponentially growing SMCs is due to residual, biologically active, mucopolysaccharide produced by the primary postconfluent SMCs from which the exponentially growing SMCs were derived. These studies suggest that bovine aortic SMCs are capable of controlling their own growth by the synthesis of a specific form of heparan sulfate with antiproliferative potency.     

Influence of macrophages and macrophage-modified collagen I on the adhesion and proliferation of vascular smooth muscle cells in culture

The adhesion, proliferation and morphology of rat vascular smooth muscle cells (VSMC) in cocultures with macrophages or in cultures on type I collagen modified by activated macrophages were evaluated. In the first set of experiments, rat alveolar macrophages were added to 24-hour-old VSMC cultures. Between days 2 and 5 after VSMC seeding, the population densities and doubling times of cells were similar in both VSMC-macrophage and pure VSMC cultures. However, from day 5, the cocultures proliferated about two times more rapidly and on day 7, they reached higher cell population density by 40%. The pure macrophage cultures did not proliferate. In the second set of experiments, rat alveolar macrophages were activated by non-toxic TiO2 dust to produce reactive oxygen species and incubated for 120 min with collagen I. The collagen was then adsorbed on plastic culture dishes and seeded with VSMC. The collagen exposed for 10 min only, the unmodified collagen and pure culture dishes were used as control growth supports. On all four tested substrates, the number of initially adhered cells was similar, but on the collagen modified for 120 min, the cells were less spread. Moreover, on day 2 to 3 after seeding, some cells on this collagen became vacuolated and detached spontaneously from the growth support. The remaining VSMC, however, rapidly proliferated, so that on day 9, the cell population density on 120-min-modified collagen was similar as on both control collagens and significantly higher compared to that on uncoated dishes. Our results suggest that 1. The delayed growth-stimulating effect of macrophages on VSMC-macrophage mixed population is probably due to autocrine production of mitogens by both cell types rather than due to an acute effect of short-living oxygen radicals released from macrophages immediately after adding to VSMC cultures. 2. The effect of collagen I exposed to activated macrophages for 120 min is slightly cytotoxic, which could, however, stimulate a release of mitogens from damaged as well as surviving VSMC.     

Platelet lysate: a replacement for fetal bovine serum in animal cell culture?

A new cell culture supplement, platelet lysate, was evaluated with reference to fetal bovine serum (FBS), an established industrial medium for animal cell culture. Chemical and bacteriological profiles were conducted including the presence of platelet-derived growth factor (PDGF). PDGF was detected in the platelet lysate but it was not present in FBS. The platelet lysate medium demonstrated lack of microorganisms, mycoplasma and endotoxins. The platelet lysate was investigated in culture studies (cell growth, viability and product formation) towards a number of target cells including myelomas, hybridomas, hepatocytes, fibroblasts and epithelial cells. In general the platelet lysate medium supported cell growth and maintained viabilities comparable or superior to fetal bovine serum. Productivity studies of antibodies (hybridomas) and transferrin (hepatocytes) showed similar or enhanced production in platelet-derived medium in comparison with FBS. This revised version was published online in July 2006 with corrections to the Cover Date.     

Stimulant-free preculture in heterologous serum-supplemented medium induces unresponsiveness of T cells to subsequent mitogenic stimulation

Quite often freshly isolated lymphocytes are kept in culture before experimentation for 1 or more days without any stimulus. Most of the time, culture is supplemented with fetal bovine serum (FBS) which is heterologous to all species except bovine. In the present study, we found that freshly isolated murine T cells show a good proliferative response to concanavalin A (Con A) and phorbol ester (PMA)/ionomycin in FBS medium, without any detectable background proliferation. However, the cells kept in the same culture without any stimulus for prolonged period of time (referred to as preculture in this report) showed reduced response to Con A and PMA/ionomycin in a time-dependent manner. Almost a complete loss of response to Con A was observed within 1 day of preculture. However, loss of response to PMA/ionomycin was observed only after 2 days of preculture. Interestingly, similar preculture in autologous mouse serum-supplemented media did not cause any loss of the response to these mitogens. The loss of responsiveness of T cells during preculture in heterologous serum was irreversible. The heterologous serum-induced unresponsiveness of T cells to these mitogens was also prevented by adding Calphostin C, a specific protein kinase C (PKC) inhibitor, during preculture in heterologous serum. These results showed that prolonged stimulant-free preculture in heterologous serum induces irreversible unresponsiveness of T cells to mitogens through the down regulation of T cell receptor signaling pathway, which can be prevented by autologous serum or a PKC inhibitor.     

Heparin selectively inhibits a protein kinase C-dependent mechanism of cell cycle progression in calf aortic smooth muscle cells [published erratum appears in J Cell Biol 1990 Mar;110(3):863]

The proliferation of arterial smooth muscle cells (SMCs) plays a critical role in the pathogenesis of arteriosclerosis. Previous studies have indicated that the glycosaminoglycan heparin specifically inhibited the growth of vascular SMCs in vivo and in culture, although the precise mechanism(s) of action have not been elucidated. In this study, we have examined the ability of specific mitogens (PDGF, EGF, heparin-binding growth factors, phorbol esters, and insulin) to stimulate SMC proliferation. Our results indicate that SMCs derived from different species and vascular sources respond differently to these growth factors. We next examined the ability of heparin to inhibit the proliferative responses to these mitogens. In calf aortic SMCs, heparin inhibits a protein kinase C-dependent pathway for mitogenesis. Detailed cell cycle analysis revealed several new features of the effects of heparin on SMCs. For example, heparin has two effects on the Go----S transition: it delays entry into S phase and also reduces the number of cells entering the cycle from Go. Using two separate experimental approaches, we found that heparin must be present during the last 4 h before S phase, suggesting a mid-to-late G1 heparin block. In addition, our data indicate that heparin-treated SMCs, while initially blocked in mid-to-late G1, slowly move back into a quiescent growth state in the continued presence of heparin. These results suggest that heparin may have multiple targets for its antiproliferative effect.     

Effects of carp serum on the growth of goldfish fin cells in early passage

The culture medium supplemented with carp serum and fetal bovine serum (FBS) promoted cell growth significantly and induced morphological change of goldfish fin cells in early passage as compared to the medium containing FBS alone. However, these effects were not observed in RBCF-1, a cell line established from the goldfish fin. The sensitivity of the cells in early passage to carp serum suggests the following possibilities: (1) cells in early passage retain the ability to respond to growth-promoting factors specifically included in carp serum; and (2) this ability is lost during the process of long-term culture and/or long-term culture in FBS eliminates cell groups showing high dependency of cell growth on carp serum.     

Establishment and conditions for growth and differentiation of a myoblast cell line derived from the semimembranosus muscle of newborn piglets

To establish an adequate model to study the proliferation and differentiation of porcine skeletal muscle in response to bioactive compounds, a pool of satellite cells was derived from the semimembranosus muscle (SM) of newborn piglets using a Percoll gradient centrifugation. The final yield amounted to 4.1 × 10⁶ cells/g muscle tissue. The percentage of muscle satellite cells has been determined by immunostaining for desmin and subsequent fluorescence analysis by flow cytometry, which revealed 95% of desmin-positive cells. For proliferation studies, satellite cell born myoblasts were seeded in gelatin-coated 96-well microplates at about 5 × 10³ cells per well. Cells were grown for 1 day in MEMα plus 10% fetal bovine serum (FBS) and 10% horse serum (HS), followed by 2 d cultivation in serum-free growth medium. For differentiation studies, myoblasts were cultured in matrigel-coated 24-well plates for 4 d with growth medium containing 10% FBS and 10% HS. At 80% confluence, cells were grown for 24 h in medium plus 10% FBS and 1 μM insulin to initiate differentiation. Subsequently, the cells were cultured in serum-free differentiation medium (SFDM) for 3 d to form myotubes. Cultures reached a maximum fusion rate of approximately 20% after 96 h. By establishing this culture system, we provide an advanced and appropriate in vitro model to study porcine skeletal muscle cell growth and differentiation including the responses to various bioactive compounds.     

In vitro study of smooth muscle cells on polycaprolactone and collagen nanofibrous matrices

Biodegradable polycaprolactone and collagen nanofibers were produced by electrospinning, with fiber diameters of around 300-700nm and features similar to the extracellular matrix of natural tissue. Human coronary artery smooth muscle cells (SMCs) seeded on nanofibrous matrices tend to maintain normal phenotypic shape and growth tends to be guided by the nanofiber orientation. The SMC and nanofibrous matrix interaction was observed by SEM, MTS assay, trypan blue exclusion method and laser scanning confocal microscopy. The results showed that the proliferation and growth rate of SMCs were not different on polycaprolactone (PCL) nanofibrous matrices coated with collagen or tissue culture plates. PCL nanofibrous matrices coated with collagen showed that the SMCs migrated towards inside the nanofibrous matrices and formed smooth muscle tissue. This approach may be useful for engineering a variety of tissues in various structures and shapes, and also to demonstrate the importance of matching both the initial mechanical properties and degradation rate of nanofibrous matrices to the specific tissue engineering.