Effect of purine supplementation on the growth of salmonid cell lines in different mammalian sera

The Chinook salmon embryo cell line, CHSE-214, grew well in fetal bovine serum (FBS) but poorly in dialyzed (d) FBS. Purines restored most but not all growth-promoting activity to dFBS, which suggests that purines account for a large portion of the dialyzable fraction's growth-promoting activity. CHSE-214 died in newborn calf serum (NCS) but grew slightly in dNCS, which suggests that the dialyzable fraction of NCS contains a toxic component(s). Little or no proliferation occurred in calf serum (CS); some took place in horse serum (HS). Porcine serum (PS) was very toxic. In all these sera except PS and HS, the purine nucleoside, inosine, significantly enhanced growth, whereas the pyrimidine nucleoside, uridine, was without effect. The other purines, hypoxanthine, adenine, adenosine and guanosine also stimulated proliferation but not as well as inosine. Inosine also enhanced the growth of the rainbow trout gonadal cell line, RTG-2. Although their morphology underwent minor alterations in medium with inosine, CHSE-214 cells could be grown indefinitely in CS and inosine as effectively as in the more expensive FBS.     

Enhancement of proliferation in cultures of Chinook salmon embryo cells by interactions between inosine and bovine sera

The influence of inosine on DNA synthesis by Chinook salmon embryo cells (CHSE-214) was investigated because previously cell number was shown to increase from six- to thirtyfold if inosine was added to the basal medium (L-15) supplemented with either dialyzed fetal bovine serum (dFBS), calf serum (CS), or dCS. Relative to L-15, ³H-thymidine incorporation was inhibited by these sera alone but elevated in nondialyzed (intact) FBS. Inosine at 10 μM stimulated ³H-thymidine incorporation from ten- to seventyfold in dFBS, CS, and dCS but was only slightly stimulatory in FBS and in L-15 alone. As well as inosine, hypoxanthine, cIMP, IMP, IDP, and ITP were just as stimulatory, but the nonsalvageable purines (xanthine, xanthosine, and XMP) were not. The stimulatory action of inosine was highest in low density cultures. Dipyridamole and S-(p-nitrobenzyl)-6-thioinosine (NBTI), inhibitors of facilitated nonconcentrative nucleoside transport, did not completely block the enhancement of cell number by inosine and by themselves increased proliferation in CS and dCS. Overall, these results suggest that exogenous inosine promoted CHSE-214 proliferation by overcoming factors in the nondialyzable fraction of sera that led to purine loss and by raising intracelular purine nucleotides to levels necessary for cells to respond to growth factors in the nondialyzable fraction of sera. © 1994 Wiley-Liss, Inc.     

Chinese hamster ovary cells cultured in low concentrations of fetal bovine serum: cloning efficiency, growth in suspension, and selection of drug-resistant mutant phenotypes

Reducing serum concentrations in media provides a potential cost advantage. To determine whether such media could be used for applied mutagenesis assays, we measured cloning efficiency and growth parameters in suspension of Chinese hamster ovary cells cultured in reduced serum with or without additives (1 microgram/ml insulin, 3 X 10(-7) M linoleic acid, 1 X 10(-8) M H2SeO3) or bovine serum albumin (BSA, 1% wt/vol). With the additives and less than or equal to 0.5% fetal bovine serum (FBS), Ham's F12 medium (without hypoxanthine and thymidine) was more optimal than alpha Eagle's minimum essential medium (MEM) (without ribosides and deoxyribosides) for low density cloning and high density suspension growth. Acceptable cloning efficiencies were obtained with 2% FBS plus BSA without additives in either medium; the addition of BSA resulted in improved colony size and more compact colony morphology. In alpha MEM, satisfactory growth rates and maximum saturation densities in suspension culture were obtained only with 5% FBS; in Ham's F12, 1% FBS + deoxycytidine + BSA yielded satisfactory suspension growth. Spontaneous mutant frequencies were compared for each medium containing 10% dialyzed FBS (DFBS), 1% FBS plus BSA, or 2% FBS plus BSA. The spontaneous frequency of azaadenine-resistant phenotypes (mutant at the aprt locus) in 1% FBS plus BSA was significantly lower than the frequency observed in 2% FBS plus BSA or 10% DFBS. Frequencies of spontaneous mutants resistant to thioguanine (hgprt locus) or fluorodeoxyuridine (tk locus) were similar with 10% DFBS, 1% FBS plus BSA, or 2% FBS plus BSA. Compared to alpha MEM with 10% DFBS, frequencies of drug-resistant mutants induced by ethyl methanesulfonate or mitomycin C (MMC) were not significantly lower in alpha MEM with 2% FBS plus BSA; observed mutant frequencies induced by dimethylnitrosamine or benzo(a)pyrene seemed to be decreased at lower survival levels.     

Endogenous thymidine and hypoxanthine are a source of error in evaluating methotrexate cytotoxicity by clonogenic assays using undialyzed fetal bovine serum

None of 13 fresh human tumor samples of various histology cloned in a two-layer agar culture system with 20% undialyzed fetal bovine serum (FBS) showed sensitivity to three antifolates, methotrexate (MTX), trimetrexate and 5,8-dideazaisofolic acid (IAHQ), even after continuous exposure to the highest concentrations (100 microM) for 21 days. In order to investigate this lack of antifolate drug effect, we compared the toxicity of continuous MTX exposure in the human colon carcinoma cell line HCT-8, cloned in a thymidineless medium (RPMI 1640) supplemented with 10% horse serum (HS), 10% fetal bovine serum (FBS), 20% FBS or 20% dialyzed FBS. In the presence of native FBS, when the minimum clone size was set at 30 cells/colony, the survival of HCT-8 cells reached a plateau at approximately 60% of untreated control after exposure to MTX concentrations between 0.1 microM and 100 microM. Only when the minimum clone size was set at 2 X 10(3) cells/colony was the sensitivity of HCT-8 cells to the antimetabolite comparable to that obtained in HS or dialyzed FBS (ED50 values in the range of 0.01 microM). MTX protection experiments indicated that even very small concentrations of thymidine and hypoxanthine together were sufficient to reproduce the pattern of sensitivity to MTX observed under culture conditions with undialyzed FBS. We conclude that for a proper evaluation of MTX cytotoxicity in clonogenic assays, dialyzed FBS and thymidine-less media should be employed; if native FBS is an absolute requirement for growth, only very large colonies (at least 10 cell divisions) should be scored.     

Chinese hamster ovary cells cultured in low concentrations of fetal bovine serum: Cloning efficiency,growth in suspension,and selection of drug-resistant mutant phenotypes

Summary Reducing serum concentrations in media provides a potential cost advantage. To determine whether such media could be used for applied mutagenesis assays, we measured cloning efficiency and growth parameters in suspension of Chinese hamster ovary cells cultured in reduced serum with or without additives (1 μg/ml insulin, 3×10⁻⁷ M linoleic acid, 1×10⁻⁸ M H₂SeO₃) or bovine serum albumin (BSA, 1% wt/vol). With the additives and ≤0.5% fetal bovine serum (FBS), Ham’s F12 medium (without hypoxanthine and thymidine) was more optimal than alpha Eagle’s minimum essential medium (MEM) (without ribosides and deoxyribosides) for low density cloning and high density suspension growth. Acceptable cloning efficiencies were obtained with 2% FBS plus BSA without additives in either medium; the addition of BSA resulted in improved colony size and more compact colony morphology. In alpha MEM, satisfactory growth rates and maximum saturation densities in suspension culture were obtained only with 5% FBS; in Ham’s F12, 1% FBS+deoxycytidine+BSA yielded satisfactory suspension growth. Spontaneous mutant frequencies were compared for each medium containing 10% dialyzed FBS (DFBS), 1% FBS plus BSA, or 2% FBS plus BSA. The spontaneous frequency of azaadenine-resistant phenotypes (mutant at theaprt locus) in 1% FBS plus BSA was significantly lower than the frequency observed in 2% FBS plus BSA or 10% DFBS. Frequencies of spontaneous mutants resistant to thioguanine (hgprt locus) or fluorodeoxyuridine (tk locus) were similar with 10% DFBS, 1% FBS plus BSA, or 2% FBS plus BSA. Compared to alpha MEM with 10% DFBS, frequencies of drug-resistant mutants induced by ethyl methanesulfonate or mitomycin C (MMC) were not significantly lower in alpha MEM with 2% FBS plus BSA; observed mutant frequencies induced by dimethyl-nitrosamine or benzo(a)pyrene seemed to be decreased at lower survival levels. This work was performed under the auspices of the U.S. Department of Energy by the Lawrence Livermore National Laboratory under Contract W-7405-ENG-48 and supported by the Environmental Protection Agency under Interagency Agreements IAG-D5-E681-AN and AO.     

Growth of human skin fibroblasts in dialyzed fetal bovine serum

Summary Human diploid fibroblast cultures plated at or below a density of 2×10³ cells per cm² grew very slowly or not at all in MEM supplemented with 10% fetal bovine serum that had been dialyzed for 24 hr. Adding serine (0.2 mM) or pyruvate (1.0 mM) to MEM and 10% dialyzed serum restored growth to the level observed with 10% nondialyzed serum. Serine and pyruvate also were able to overcome partially the growth arrest induced by a reduced serum concentration (1 or 2%). Human fibroblast cultures grew very well in 100% fetal bovine serum that had been dialyzed against MEM. For cells grown in dialyzed serum, the final number increased with increasing serum concentration, in contrast to the well established toxic effects of high concentrations of nondialyzed serum. This research was supported by NIH Grants CA15207 and HD03110.     

The influence of zinc status on the kinetics of zinc uptake into cultured endothelial cells

To better understand cellular zinc homeostasis and characterize the zinc transport process, a mammalian cell culture model was utilized to investigate the influence of zinc status on the kinetics of zinc uptake. Culturing conditions were optimized to induce moderate zinc deficiency and zinc excess while still sustaining the general health of the cells. Cells were grown in (1) control medium of 10% fetal bovine serum (FBS) in minimum essential medium (MEM; 5.0 micromol zinc/L), (2) low zinc medium (10% dialyzed FBS in MEM; 1.5 micromol zinc/L), or (3) zinc back medium (10% dialyzed FBS in MEM with zinc added as ZnCl(2); 5.0 micromol zinc/L). Bovine pulmonary artery endothelial cells (BPAEC), porcine aortic endothelial cells (PAEC), and porcine venous endothelial cells (PVEC) were evaluated as to their responsiveness to our zinc-deficient conditions. Zinc uptake was faster (P < 0.001) in all three cell types when they were grown in low zinc medium compared with controls; the increases were 32% in PAEC, 37% in PVEC, and 66% in BPAEC. Further kinetic analysis with BPAEC demonstrated a 31% increase (P < 0.05) in the maximum rate of zinc uptake (Jmax) grown in low zinc medium compared with controls, but no difference (P > 0.05) between the low zinc group and the control group in the concentration at which uptake was half-maximal (K). Zinc uptake into BPAEC grown in excess zinc conditions was not different (P > 0.05) unless the medium contained greater than 50 micromol zinc/L. In conclusion, BPAEC increased their ability for zinc uptake in response to moderate zinc deficiency, but did not change their kinetics of zinc uptake during moderate zinc excess.     

Growth of fish cell lines in glutamine-free media

The glutamine requirement for thein vitro proliferation of fish cells was investigated with cell lines from four different species and three tissues: goldfish skin (GFSk-S1), Chinook salmon embryo (CHSE-214), and raibow trout liver (RTL-W1) and spleen (RTSp-W1). With a supplement of fetal bovine serum, the basal medium, Leibovitz's L-15, without glutamine supported the proliferation of all four cell lines as well, or nearly as well, as L-15 with 2 mM glutamine. This was true over short term assays of two to four weeks and for continuous propagation. CHSE-214 also grew as well with or without 2 mM glutamine in Minimum Essential Medium with fetal bovine serum. However, when the supplement was dialyzed fetal bovine serum, CHSE-214 grew much better in L-15 without glutamine. Therefore, glutamine was not required for growth in L-15, and in fact, was inhibitory in the absence of the dialyzable fraction of serum. By contrast, glutamine appeared to be important for growth in Minimum Essential Medium. When the supplement was dialyzed fetal bovine serum, CHSE-214 grew much better in Minimum Essential Medium with 2 mM glutamine. These results suggest that the glutamine requirement for thein vitro proliferation of fish cells is conditional and depends on the basal medium and serum supplement.Abbreviations BSA bovine serum albumin - CHSE-214 Chinook samon embryo cell line - dFBS dialyzed fetal bovine serum - FBS fetal bovine serum - GFSk-S1 goldfish skin cell line - GS glutamine synthetase - L-15 Leibovitz's L-15 media - L929 mouse fibroblast cell line - MEM minimum essential medium Eagle - PBS phosphate buffered saline - RTL-W1 rainbow trout liver cell line - RTSp-W1 rainbow trout spleen cell line     

Mitogenic activity of fetal bovine serum, fish fry extract, insulin-like growth factor-I, and fibroblast growth factor on brown bullhead catfish cells--BB line

Bioassays were performed to assess the effects of different levels of growth medium supplementation with fetal bovine serum (FBS), fish fry extract (FE), combinations of FBS and FE, and addition of insulin-like growth factor I (IGF-I) and fibroblast growth factor (FGF) on the proliferation of brown bullhead catfish cells (BB line). Treatments (n = 4) were: 2.5, 5, 10, and 15.0% FBS or FE and 5/2.5, 5/5, 10/2.5, and 10/5 of a FBS/FE combination as supplement to the growth medium, or the addition of 0.1, 1, 2.5, 10, 25, and 75 ng/ml of either IGF-I or FGF to the growth media. Initial cell density was 1.1 x 10(6) cells per well on uncoated 24-well plates. Incubation temperature was 29.5 +/- 0.7 degrees C. Six hours after plating, initial culture medium was removed, plates rinsed with Dulbecco's phosphate buffered saline, treatment media added, and cells allowed to proliferate for 24 hours. Another bioassay was performed with rat myoblast omega cells (RMo) using the same levels of growth medium supplemented with FBS, FE and FBS/FE. Base growth medium was Dulbecco's MEM. The initial cell density was 7.2 x 10(6) cells per well, and the bioassay was carried out at 36.0 +/- 0.5 degrees C, on a 95% air, 5% CO2 incubator. Increasing levels of FBS had a positive effect (P < 0.05) on the proliferation of both BB and RMo cells. Increasing levels of FE had a negative effect (P < 0.05) on the proliferation of BB cells and totally inhibited the proliferation of RMo cells at any level of supplementation. Higher levels of FE on the FBS/FE combinations presented a negative effect on the proliferation of both BB and RMo cells (P < 0.05). Insulin-like growth factor I had a positive quadratic effect (P < 0.05) on the proliferation of BB cells. Apparently, mammalian growth factors slightly stimulated mitogenic activity in fish cells, while FE contained factors which inhibited the mitogenic activity of RMo and BB cell lines.     

The influence of serum components on the growth and mutation of Chinese hamster cells in medium containing aminopterin

When seeded in small numbers in medium containing 10^?6M aminopterin and fetal calf serum, V₇₉ Chinese hamster cells required dialyzable components from the serum for growth. However, the cells grew in medium containing 10^?6M aminopterin and dialyzed serum, provided that the medium was supplemented with 10^?5M hypoxanthine and sufficient 5·10^?6M) thymidine. A growth-inhibitory property of some batches of dialyzed serum was abolished on heating the serum for 30 min at 56°. Three lines of V₇₉ cells which lacked detectable hypoxanthine-guanine phosphoribosyl transferase (HGPRT) activity were seleccted in medium containing 8-azaguanine (8-AzG). In two of these, no spontaneous reversion to the HGPRT⁺ phenotype was detectable, and these cells did not cooperate metabolically with HGPRT⁺ cells to prevent the growth of the latter in HAT medium. One of the HGPRT^? lines showed a high rate of spontaneous reversion (118/10⁵ cells) in medium containing undialyzed serum. However, in medium containing dialyzed serum the spontaneous reversion rate fell to $\text{4}\text{10}{}^5\text{cells}$, suggesting that the revertants arising in medium containing undialyzed serum were biochemically heterogeneous.     

Effects of epidermal growth factor and transforming growth factor-beta 1 on rat heart endothelial cell anchorage-dependent and -independent growth

This report describes the effects of epidermal growth factor (EGF) and transforming growth factor-beta 1 (TGF-beta 1) on the anchorage-dependent and -independent growth of rat heart endothelial cells (RHE-1A). When RHE-1A cells were grown in monolayer culture with medium containing 10% fetal bovine serum (FBS) supplemented with epidermal growth factor (0.1-100 ng/ml), growth was stimulated fivefold when compared to that of cells grown in medium containing 10% FBS alone. The stimulatory effect of EGF on RHE-1A cell monolayer growth was dose-dependent and half-maximal at 5 ng/ml. The addition of TGF-beta 1 in the range 0.1-10 ng/ml had no effect on RHE-1A cell monolayer growth when added to medium containing 10% FBS alone or 10% FBS supplemented with EGF (50 ng/ml). RHE-1A cells failed to grow under anchorage-independent conditions in 0.3% agar medium containing 10% FBS. In the presence of EGF, however, colony formation increased dramatically. The stimulatory effect of EGF was dose-dependent in the range 0.1-100 ng/ml and was half-maximal at 5 ng/ml. In contrast to its effects under anchorage-dependent conditions, TGF-beta 1 (0.1-10 ng/ml) antagonized the stimulatory effects of EGF on RHE-1A cell anchorage-independent growth. The inhibitory effect of TGF-beta 1 was dose-dependent and half-maximal at 0.1 ng/ml. EGF-induced RHE-1A soft agar colonies were isolated and reinitiated in monolayer culture. They retained the cobblestone morphology and contact-inhibition characteristic of normal vascular endothelial cells. Each of the clones continued to express Factor VIII antigen. These findings suggest that TGF-beta may influence not only endothelial cell proliferation but also anchorage dependence. These effects may in turn be of relevance to endothelial cell growth and angiogenesis in vivo.     

Serum Can Overcome Contact Inhibition in Confluent Human Pulmonary Artery Smooth Muscle Cells

Pulmonary artery endothelial cells (PAEC) in an intact vessel are continually exposed to serum, but unless injured, do not proliferate, constrained by confluence. In contrast, pulmonary artery smooth muscle cells (PASMC) attain, and maintain, confluence in the presence of minimal serum, protected from serum’s stimulatory effects except when the endothelial barrier becomes more permeable. We hypothesized therefore, that confluent PASMC may be less constrained by contact inhibition in the presence of serum than PAEC and tested this idea by exposing confluent non-transformed human PAEC and PASMC to media containing increasing concentrations of fetal bovine serum (FBS) and determining cell growth over 7 days. PAEC that had attained confluence in low serum did not proliferate even when exposed to 5% serum, the highest concentration tested. In contrast, PASMC that attained confluence in low serum did proliferate once serum levels were increased, an effect that was dose dependent. Consistent with this observation, PASMC had more BrdU incorporation and a greater percentage of cells in S phase in 5% compared to 0.2% FBS, whereas no such difference was seen in PAEC. These results suggest that confluent human PAEC are resistant to the stimulatory effects of serum, whereas confluent PASMC can proliferate when serum levels are increased, an effect mediated in part by differences in phosphoinositide 3-kinase activation. This observation may be relevant to understanding the PASMC hyperplasia observed in humans and animals with pulmonary hypertension in which changes in endothelial permeability due to hypoxia or injury expose the underlying smooth muscle to serum.     

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.     

Expression of the fetal-oncogenic fibroblast growth factor-8/17/18 subfamily in human hematopoietic tumors

The fibroblast growth factors (FGFs) are involved in hematopoiesis and tumorigenesis. However, little is known about the contribution of the FGFs identified within the past 10 years to leukemogenesis. To elucidate whether these FGFs (FGF-8, -9, -10, -11, -12, -13, -14, -16, -17, -18, -19, -20, and -21) are expressed in leukemic cells, we performed RT-PCR analyses using 28 cell lines. The members of a fetal-oncogenic subfamily, FGF-8/-17/-18, were often expressed (53.5%, 25.0%, and 32.1%) with the co-expression of their receptors. Realtime quantitative-PCR analysis showed that FGF-8/-17 were aberrantly expressed in patients with acute leukemia. Moreover, cell proliferation assays revealed the proliferation activity of FGF-17 on leukemic cells expressing its receptors. These results demonstrated that certain recently identified FGFs play an important role in the growth of leukemic cells, possibly with an autocrine mode of action, and that these FGFs will become novel biomarkers for hematopoietic tumors.     

Nanofiltration of growth media supplemented with human platelet lysates for pathogen-safe xeno-free expansion of mesenchymal stromal cells

Background aimsHuman platelet lysate can replace fetal bovine serum (FBS) for xeno-free ex vivo expansion of mesenchymal stromal cells (MSCs), but pooling of platelet concentrates (PCs) increases risks of pathogen transmission. We evaluated the feasibility of performing nanofiltration of platelet lysates and determined the impact on expansion of bone marrow–derived MSCs.MethodsPlatelet lysates were prepared by freeze-thawing of pathogen-reduced (Intercept) PCs suspended in 65% storage solution (SPP+) and 35% plasma, and by serum-conversion of PCs suspended in 100% plasma. Lysates were added to the MSC growth media at 10% (v/v), filtered and subjected to cascade nanofiltration on 35- and 19-nm Planova filters. Media supplemented with 10% starting platelet lysates or FBS were used as the controls. Impacts of nanofiltration on the growth media composition, removal of platelet extracellular vesicles (PEVs) and MSC expansion were evaluated.ResultsNanofiltration did not detrimentally affect contents of total protein and growth factors or the biochemical composition. The clearance factor of PEVs was >3 log values. Expansion, proliferation, membrane markers, differentiation potential and immunosuppressive properties of cells in nanofiltered media were consistently better than those expanded in FBS-supplemented media. Compared with FBS, chondrogenesis and osteogenesis genes were expressed more in nanofiltered media, and there were fewer senescent cells over six passages.ConclusionsNanofiltration of growth media supplemented with two types of platelet lysates, including one prepared from pathogen-reduced PCs, is technically feasible. These data support the possibility of developing pathogen-reduced xeno-free growth media for clinical-grade propagation of human cells.     

Effect of different mitogens and serum concentration on HUVEC morphology and characteristics: implication on use of higher passage cells

Human umbilical vein endothelial cells (HUVEC) were cultured in two different media, viz. the commonly used M199 containing 20% fetal bovine serum (FBS) and endothelial cell growth factor and a defined media EGM-2 containing 2% FBS along with growth supplements in known concentrations. The purpose of this study was to determine the effect of different media on the growth potential and cell morphology in subsequent passages.We have established that a dual coating of gelatin and human fibronectin extracellular matrix provides optimal cell attachment. Growth rate for primary culture was almost double in defined media. For secondary culture a two fold higher proliferation rate was observed in defined EGM-2 media. Histological studies were done using phase contrast, confocal and scanning electron microscopy which showed that cells cultured in M199 started losing their morphological characteristic from 3rd passage and after 6th passage appeared to come in senescent stage, while in case of defined media there was no change observed in the cells up to 10th passage. A significant difference was found in the expression of soluble intracellular adhesion molecule-1 (sICAM-1) which is an endothelial cell marker on cells cultured in different media. Additionally it was observed that exposure duration to trypsin-EDTA during cell detachment also plays an important role in maintaining cell morphological characteristics.These results show that significant morphological changes appear in higher order passages if cells are grown in routine medium for a long time and therefore may not be suitable for cell signaling experiments.     

Effects of serum fractions on the growth of mononuclear phagocytes

The effects of serum fractions on the growth kinetics and colony formation of mononuclear phagocytes derived from mouse bone marrow, blood, and peritoneal cavity were investigated. Peritoneal exudate macrophages and blood monocytes required a factor(s) found to reside in the nondialyzable serum fraction (molecular weight > 12,000) to survive, a small molecular weight (< 307) factor(s) with growth-stimulatory activity (GSA) contained in the dialyzable serum fraction, and the macrophage growth factor (MGF) for proliferation and colony formation. Fetuin, a major protein of fetal serum, was able to substitute the non-dialyzable serum fraction. Macrophages cultured in medium containing MGF and the nondialyzable serum fraction for 6 days could be restored to full growth following the addition of the dialyzable serum fraction. In contrast, bone marrow mononuclear phagocytes cultured in the absence of the dialyzable serum fraction were capable of proliferating, though at a slower rate, and forming colonies. In addition, neither insulin nor hydrocortisone was capable of replacing the serum-dialyzable GSA nor able to enhance colony formation.     

The influence of conditioned media and nonessential amino acid supplementation on the growth of cells in vitro

The growth enhancing effect of media conditioned by cells from established lines (BHK and L60) is comparable to that of media conditioned by cells of primary origin (chick embryo). However, the properties of the conditioned media from these two systems show marked differences: the growth enhancing factors in the former are dialyzable and heat-stable, in contrast to the non-dialyzable and heat-labile factors in the latter. Media conditioned for only four hours by BHK or L60 cells stimulated cell growth. Amino acid analyses revealed that non-essential amino acids had appeared in these conditioned media. To verify this as the metabolic basis of conditioning by cells from established lines, media containing dialyzed serum were supplemented with each of six non-essential amino acids, and assayed on BHK and L60 at various population densities. Serine was the most stimulatory and alanine the most inhibitory of the amino acids tested. Mixed supplementation of the medium showed that when low levels of alanine and serine were added simultaneously, cell growth was enhanced but any increase in the level of alanine required an increase in the level of serine also to achieve growth stimulation.     

海藻酸微囊替代DMSO和FBS的STO细胞冷冻保存研穷

目的：改进现有的细胞冷冻保存方法,建立一个不舍二甲基亚砜（DMSO）和血清（FBS）的高效冷冻保存方法,为细胞治疗等临床实践提供优质细胞。方法：海藻酸微囊包埋鼠胚成纤维细胞（STO细胞）后用不含DMSO和FBS的冷冻保存液进行冷冻保存。,设四个对照组：添加10％DMSO和20％FBS的组、仅添加10％DMSO的组、仅添加20％FBS、DMSO和FBS均不添加组。在冷冻前后对各实验组细胞用台盼兰染色,进行细胞计数,计算细胞存活率,同时利用溴乙锭的二聚物（EthD）、钙黄绿素-AM（Calcein—AM）进行染色观察细胞的形态,且进一步验证细胞存活率;解冻复苏后用MTT法评估细胞的增殖速度和生长活力。结果：冷冻保存30天后对各组的细胞数量、细胞存活率、细胞形态和解冻复苏后细胞的生长活力进行比较发现,海藻酸微囊包埋冷冻组的细胞数、细胞存活率、细胞形态和生长活力均与添加DMSO和FBS的组之间无显著性差异,而与其它三个对照组呈显著性差异。结论：使用海藻酸微囊替代DMSO和FBS保存STO细胞,能有效的维持细胞形态、数量、存活率,同时不影响细胞的生长活力,从而建立了一个不含DMS0和FBS的高效冷冻保存方法。