In vitro Proliferation of Human Bone Marrow Mesenchymal Stem Cells Employing Donor Serum and Basic Fibroblast Growth Factor

A method for the in vitro proliferation of human bone marrow mesenchymal stem cells (MSCs) employing a medium not containing fetal calf serum (FCS) was developed for a regenerative medicine of cartilage using MSCs. Without using density-gradient centrifugation, the bone marrow aspirate was poured into a dish (6.0 \times 10⁵ nucleated cells/cm²) with DMEM medium containing 10% serum (FCS or donor serum) and basic fibroblast growth factor, and incubated at 37 °C under a 5% CO₂ atmosphere. The density of adhesive cells incubated with the medium containing human serum and basic fibroblast growth factor (10 ng/ml) almost reached confluence at 19d and was 1.4-2.7 times that in the medium containing only FCS. The density of cells incubated with the medium containing only human serum was 0.1-0.6 times that in the medium containing only FCS. The content of CD45^- CD105⁺ cells among the cells harvested after a 19-d incubation in the medium containing human serum and basic fibroblast growth factor was higher than 90%. This high content and chondrogenic activity, which was confirmed by pellet cultivation and staining with Safranine O, were maintained even after further subcultivation in the medium to 17 population doubling levels. Consequently, this method might be applicable to in vitro proliferation of MSCs for the regeneration of cartilage.     

Trypanosoma brucei gambiense: HMI-9 medium containing methylcellulose and human serum supports the continuous axenic in vitro propagation of the bloodstream form

Trypanosoma brucei (T.b.) gambiense causes the chronic form of human African trypanosomiasis or sleeping sickness. One of the major problems with studying T.b. gambiense is the difficulty to isolate it from its original host and the difficult adaptation to in vivo and in vitro mass propagation. The objective of this study was to evaluate if an established method for axenic culture of pleomorphic bloodstream form T.b. brucei strains, based on methylcellulose containing HMI-9 medium, also facilitated the continuous in vitro propagation of other bloodstream form Trypanozoon strains, in particular of T.b. gambiense. Bloodstream form trypanosomes from one T.b. brucei, two T.b. rhodesiense, one T. evansi and seven T.b. gambiense strains were isolated from mouse blood and each was concurrently cultivated in liquid and methylcellulose-containing HMI-9 based medium, either with or without additional human serum supplementation, for over 10 consecutive sub passages. Although HMI-9 based medium supplemented with 1.1% (w/v) methylcellulose supported the continuous cultivation of all non-gambiense strains better than liquid media could, the in vitro cultivation of all gambiense strains was only achieved in HMI-9 based medium containing 1.1% (w/v) methylcellulose, 15% (v/v) fetal calf serum and 5% (v/v) heat-inactivated human serum.     

Effects of defined medium,fetal bovine serum,and human serum on growth and chemosensitivities of human breast cancer cells in primary culture: Inference for in vitro assays

Summary We compared the effects of defined medium, fetal bovine serum (FBS) and human serum (HuS) on the growth and responses to chemotherapeutic agents of human breast cancer cells in primary culture. Normal and tumor tissues were dissociated to small aggregates and single cells and seeded onto collagen-gel-coated wells in defined medium or medium supplemented with 5% FBS or 5% HuS. In all cases examined, defined medium and medium containing HuS were superior to medium containing FBS in supporting growth of both normal and tumor cell cultures. However, cultures in defined medium showed an initial cell loss. Cells from the same tumor cultured in different media varied in their responses to chemotherapeutic agents. In light of these results, medium supplemented with HuS, which promoted attachment of these cells in culture and stimulated their growth, should be the most appropriate nutrient environment for determining the effects of therapeutic agents on cells as it most closely resembles the in vivo situation. Because there were also variations in growth rates and chemosensitivities of tumor cells cultured in different human serum samples, we suggest that optimal conditions in which to culture these cells include the serum of the patient whose tumor is removed. This serum may provide host factors that influence cell growth and interact with exogenous factors. This work was supported by a grant from the National Cancer Institute of Canada and funds contributed by Mr. B. T. Wharton in memory of his wife, Nadia. J. T. Emerman is a research scholar of the National Cancer Institute of Canada.     

Secretion of alpha-L-fucosidase by human embryonal skin fibroblasts

The amount of alpha-L-fucosidase secreted by normal human fibroblasts was higher in the medium containing 10% bovine serum than in the medium containing 0.1% bovine serum. Glycosidase secretion was twice higher at the advanced than at the initial stage of subcultivation. Extracellular activity of alpha-L-fucosidase from 3 different fibroblast strains differed insignificantly in the medium containing 0.1% bovine serum, while intracellular activity of the enzyme in these strains was altogether different. The results suggest that the lysosomal glycosidase secretion is determined by the level of cellular endocytosis.     

Partial purification of human lymphocyte activating factor

Lymphocyte Activating Factor (LAF) is a T lymphocyte stimulant released by human monocytes cultured for 18-24 hours in tissue culture medium containing 5% human serum and the non-specific immunostimulant lipopolysaccharide. The purification of LAF is essentially the separation of low MW LAF (approximately 13,000) from the human serum proteins required for production of the activity. Hollow fiber ultrafiltration has been found to effect a rapid separation of low MW LAF from serum proteins, but with a yield of only 20% of the original activity. Isoelectric focusing (IEF) efficiently separates LAF from all traces of human serum, resulting in a purified sample containing no measurable protein and revealing no bands on polyacrylamide gels. The IEF purified material is about 2% of the low MW activity present in the unfractionated culture medium and is highly active in the biological assay system.     

Partial Purification of Human Lymphocyte Activating Factor

Lymphocyte Activating Factor (LAF) is a T lymphocyte stimulant released by human monocytes cultured for 18–24 hours in tissue culture medium containing 5% human serum and the non-specific immunostimulant lipopoly-saccharide. The purification of LAF is essentially the separation of low MW LAF (～13.000) from the human serum proteins required for production of the activity. Hollow fiber ultrafiltration has been found to effect a rapid separation of low MW LAF from serum proteins, but with a yield of only 20% of the original activity. Isoelectric focusing (IEF) efficiently separates LAF from all traces of human serum, resulting in a purified sample containing no measurable protein and revealing no bands on polyacrylamide gels. The IEF purified material is about 2% of the low MW activity present in the unfractionated culture medium and is highly active in the biological assay system.     

Effects of subcultivation and culture medium on differentiation of human fetal cardiac myocytes

Summary Previous work has suggested that subcultivated human fetal heart muscle cell cultures contain immature cardiac muscle cells capable only of limited differentiation after mitogen withdrawal. We studied several human fetal heart cultures (14–15 wk gestation) at several passage levels using immunocytochemistry, autoradiography, and Northern blot analysis. Characteristics in high-mitogen (growth) medium were compared with those after serum withdrawal. Cultured cells from one heart, expanded through 2 passages in growth medium, did not beat; however, 75% of cells did beat after subsequent culture for 24 days in low-serum (differentiation) medium containing insulin. In confluent cultures after 1 passage, there was no detectable difference in the number of cardiac myocytes present in growth medium compared with that 7 days after serum withdrawal. After 4 passages, however, serum withdrawal increased the number of cells expressing immunoreactive sarcomeric myosin heavy chain by 100-fold; expression of immunoreactive sarcomeric actin andα-cardiac actin mRNA also increased in the same cultures. Similar results were obtained in cultures kept in differentiation medium for 20 days before passage and expansion in growth medium. Using isopycinc centrifugation, a high-density cell fraction was isolated which contained no immunostained myocytes in growth medium but numerous myocytes after serum withdrawal. Combined immunocytochemistry/autoradiography showed that myocytes synthesize DNA in growth medium and in serum-free medium containing fibroblast growth factor, but not in serum-free medium alone. The results indicate that a) human fetal cardiac muscle cells proliferate in vitro and can maintain a phenotype characteristic of fetal myocytes after multiple subcultivations followed by serum withdrawal; b) after subcultivation in growth medium, some myocytes modulate their phenotype into one in which detectable levels of cardiac contractile proteins are expressed only after mitogen withdrawal, and c) the phenotype attained after serum withdrawal is in part dependent on passage level. Cultured human fetal myocardial cells my provide a useful experimental system for the study of human cardiac muscle cell biology.     

Purification and characterisation of cell survival factor 1 (TCSF1) from Tetrahymena thermophila

Of a number of peptides isolated from the extracellular medium of Tetrahymena cultures, two with masses 9.9 and 22.4 kDa allowed low-density cultures of this ciliate to survive and enter a proliferate phase. The smaller peptide (TCSF1) also greatly helped cultured mammalian fibroblasts to survive in medium containing very low concentrations of serum for considerably longer than controls, and to grow when full strength medium was restored. The primary sequence of the TCSF1 was determined, and synthetic TCSF1 was observed to exhibit rescuing activity comparable to that of the native peptide.     

The formation of stable E-rosettes by human T lymphocytes activated in mixed lymphocyte reactions.

The aim of the present study was to determine whether activation of human T-lymphocytes affects their interaction with sheep red blood cells (SRBC). Less than 3% of the E-rosettes formed by freshly isolated peripheral blood lymphocytes (PBL) and SRBC are stable and do not disintegrate after incubation at 37 degrees C. In contrast, about 30% of PBL kept in culture for 5 days in the presence of mitomycin C-treated allogeneic lymphocytes were found to form stable E-rosettes. Whereas no rosettes were formed by freshly isolated PBL incubated with human red blood cells at 24 degrees C, 15% of the cells recovered from mixed lymphocyte reactions (MLR) formed such rosettes. When responder PBL were maintained in culture in the absence of allogeneic stimuli the proportion of cells forming stable E-rosettes depended on the serum present in the medium. Less than 5% of the responder cells kept in medium containing human serum or in serum-free medium formed stable E-rosettes, whereas 18% of the cells maintained in medium containing fetal calf serum formed stable E-rosettes. The proportion of cells forming stable E-rosettes increased before any increase in DNA synthesis was detectable in MLR. Indeed, a high proportion of cells forming stable E-rosettes appeared in MLR taking place in serum-free medium, without any accompanying increase of DNA synthesis. Depletion of cells forming EAC'-rosettes from responder PBL increased the proportion of cells forming stable E-rosettes in MLR. Exposure of the cells recovered in MLR to specific anti-T sera inhibited the formation of both stable and regular E-rosettes. Exposure of the cells recovered in MLR to anti-Ig serum had no effect on the formation of regular rosettes. Anti-Ig serum strongly inhibited the formation of stable E-rosettes by cells grown in medium containing human serum, but had no effect on the formation of stable E-rossettes by cells grown in either serum-free medium or in serum containing fetal calf serum. It is concluded that activated human T lymphocytes are characterized by their capacity to form stable E-rosettes, resistant to incubation at 37 degrees C, and by their capacity to acquire an immunoglobulin coat, possibly by binding immunoglobulin molecules present in their environment.     

High-density lipoprotein prevents SAA-induced production of TNF-α in THP-1 monocytic cells and peripheral blood mononuclear cells

In this study, we evaluated whether human serum and lipoproteins, especially high-density lipoprotein (HDL), affected serum amyloid A (SAA)-induced cytokine release. We verified the effects of SAA on THP-1 cells in serum-free medium compared to medium containing human serum or lipoprotein-deficient serum. SAA-induced tumour necrosis factor-alpha (TNF-α) production was higher in the medium containing lipoprotein-deficient serum than in the medium containing normal human serum. The addition of HDL inhibited the SAA-induced TNF-α release in a dose-dependent manner. This inhibitory effect was specific for HDL and was not affected by low-density lipoprotein or very low-density lipoprotein. In human peripheral blood mononuclear cells, the inhibitory effect of HDL on TNF-α production induced by SAA was less pronounced. However, this effect was significant when HDL was added to lipoprotein-deficient medium. In addition, a similar inhibitory effect was observed for interleukin-1 beta release. These findings confirm the important role of HDL and support our previous hypothesis that HDL inhibits the effects of SAA during SAA transport in the bloodstream. Moreover, the HDL-induced reduction in the proinflammatory activity of SAA emphasizes the involvement of SAA in diseases, such as atherosclerosis, that are characterized by low levels of HDL.     

Improved media for normal human muscle satellite cells: Serum-free clonal growth and enhanced growth with low serum

Summary We have developed a serum-free medium for clonal growth of normal human muscle satellite cells (HMSC). It consists of an optimized nutrient medium MCDB 120, plus a serum-free supplement, designated SF, that contains epidermal growth factor (EGF), insulin, dexamethasone, bovine serum albumin, and fetuin. Fibroblast growth factor was needed with dialyzed fetal bovine serum (dFBS) as the only other supplement, but in media containing SF, it was only slightly beneficial, and was omitted from the final medium without significant loss. Clonal growth of HMSC in MCDB 120 plus SF is as good as with 15% serum and 0.5% chicken embryo or bovine pituitary extract. However, growth is further improved by use of a doubly-supplemented (DS) medium containing both SF and 5% dFBS. Clonal growth of HMSC in the DS medium far exceeds that in previous media with any amount of serum, and monolayer growth is at least equal to that in conventional media with higher levels of serum. Cells grown in these media exhibit little differentiation, even when grown to high densities. However, they retain the capacity for extensive fusion and synthesis of increased creatine kinase when transferred to a serum-free differentiation-promoting medium, such as Dulbecco's modified Eagle's medium plus insulin. All experiments were done with clonal cultures of HMSC to insure that observed growth responses were always those of muscle cells. This research was supported by a grant from the Muscular, Dystrophy Association. Editor's statement This article describes the optimization of both the basal nutrient medium and growth factor requirements for human muscle cells in vitro. This system is critical for studies of normal muscle cell and molecular biology, as well as for understanding diseases of muscle such as Duchenne, Muscular Dystrophy.     

Primary culture and maintenance of steroid-secreting human placenatal monolayers.

A simple method is described for primary culture and for maintenance of hormone-producing cells from normal human placenta. A consistent yield of cells was obtained and an average survival of 3 to 4 months in culture using 1 mm3 explants from the most vascular area of the placentas. These explants were placed in a variety of culture media in 30 ml flasks and incubated at 37 degrees C in an atmosphere of 5% CO2 and 95% air. The best yields in terms of cell growth were observed with Eagle's MEM (minimum essential medium) with supplements of horse serum and fetal calf serum or human cord serum. (Ham's F-10 with supplement of horse serum and fetal calf serum supports growth for the longest period and media containing human cord serum had the best yield of steroids.     

Reduced frequency of baseline sister chromatid exchanges in lymphocytes grown in antibiotics and serum-excluded culture medium

Peripheral venous blood from man, muntjac, and cattle were grown in medium (1) containing different serum (human AB+/FCS/autologous), (2) without serum or antibiotics (penicillin and streptomycin), or (3) without both serum and antibiotics to investigate to what extent certain essential culture components used in in vitro mammalian cell cultures affect the incidence of spontaneous sister chromatid exchanges (SCEs). The observation that exclusion of only serum from culture medium enhanced the frequency of SCEs whereas exclusion of both serum and antibiotics, which influence the cell cycle kinetics to a great extent, exhibited the lowest frequency of SCEs reported so far for lymphocyte cultures, indicates that the frequency of so-called spontaneous SCEs usually observed in normal lymphocyte cultures grown in medium supplemented with serum and antibiotics is in fact an elevated frequency.     

Natural antibodies as contaminants of hybridoma products

This report cites an example of natural antibodies in mouse serum and ascites fluid which may contaminate hybridoma products and cause difficulties in interpreting their reactions. These natural antibodies react with determinants expressed on human foetal glycoproteins (extracted from meconium) and on other blood group precursor-like substances which express a number of tumour associated- and differentiation antigens. Variable amounts of these antibodies may be present in the ascites fluid of mice obtained by intraperitoneal injection of cloned hydridomas. Although in most cases the hybridoma antibodies will be used at dilutions beyond the endpoint of these contaminating antibodies, it is important to be aware of possible reactions due to natural and acquired antibodies with diverse specificities in evaluating the reactions of hybridoma products harvested from any type of serum containing medium.     

Kinetics of producing pro-urokinase in perfusion cultivations

Summary Better production of pro-urokinase from human cell line was observed with 5% serum containing medium than 10% or serum free medium on Cytodex II under perfusion chemostat operations, showing 0.8×10^–5 (IU/daycell) of maximum productivity at 0.020 (l/h) of dilution rate in 5% serum medium, which corresponds to 800 IU/mL at this dilution rate. Conversion of pro-urokinase was reduced in the serum-containing media.     

Altered activity in cultured cells caused by contaminants in tubes widely used for blood collection and serum preparation

Summary Cell culture has been recognized as an extremely sensitive system for measuring the toxicity of various materials. A study was done to determine whether the type of tube used to collect blood or store human serum might affect results in experiments requiring blood drawn into such tubes. In order to test tubes for contaminants that might alter cellular activity, a variety of commercially available tubes used for collection of blood and storage of serum were shaken while containing culture medium with fetal bovine serum. The medium was then applied to 3T3 fibroblasts in culture. Measuring incorporation of tritiated thymidine into DNA in log phase cells as an index of cellular proliferation, it was found that medium containing serum preincubated in tubes routinely used for blood collection could be extremely toxic. The same types of tube were also used to prepare human serum. When serum from some of the tubes was applied to 3T3 fibroblasts, a stimulatory effect was observed, perhaps caused by selective adsorption of inhibitory components of the blood or serum by various tubes. It is, therefore, crucial in a properly controlled experiment using serum in vitro to collect blood in tubes that exert no toxic or stimulatory effects in the assay or, at least, to be consistent in one’s choice of tube. None of the tubes used for storage of serum showed significant effects in our assay.     

Doubling potential, calendar time, and donor age of human diploid cells in culture

Human diploid fibroblasts from an embryonic and an adult donor were maintained in an essentially nonmitotic state for extended periods of time by reducing the serum concentration of the growth medium from 10 to 0.5%. These cells could be returned to the rapidly proliferating state by subcultivation with medium containing 10% serum. Cells treated in such a manner took a proportionately longer calendar time to reach phase III than did controls that had been continuously cultured on growth medium. The division potential of cells from the adult donor was unaffected by the arrested state; however, that of cells from the embryonic donor was extended beyond that of growth controls. The extension of division potential in cells from the embryonic donor never exceeded the maximum passage level reported for these cells. During 21 days of cultivation with medium containing 0.5% serum, cell losses ranged from 15 to 35% and the protein content of the remaining cells decreased by 15 to 20%. It was concluded that division potential and not total calendar time was the primary determinant of the in vitro lifespan of these human diploid cells and that arresting cells from younger donors increased their ability to attain the maximum limit of their lifespan.     

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.     

A low-serum medium with BSA and ferrous sulfate for the production of tissue plasminogen activator by human embryo lung cells

A low-serum medium containing bovine serum albumin (BSA) was investigated with respect to the growth of and tissue plasminogen activator (TPA) production by human embryo lung (HEL) cells on microcarrier beads and in collagen gel. BSA and ferrous sulfate were chosen as substitutes for fetal calf serum (FCS) through a simple screening test involving many substances. The growth promoting effects of BSA and ferrous sulfate were independent of each other and from the FCS concentration. Though BSA inhibited initial cell attachment to the carrier surface, it did promote the growth of cells attached to microcarrier beads. Cells grown on microcarrier beads in the low-serum medium containing BSA, ferrous sulfate and 3% FCS produced an amount of TPA similar to that produced by ones grown in the 10% FCS medium. Although cells on the dish surface did not grow at all on serum-free media containing BSA and ferrous sulfate, cells in the collagen gel were able to grow slightly on the serum-free medium. Cells grown on the low-serum medium in collagen gel produced more TPA over a long period than those in the microcarrier beads using the low-serum medium. The optimum concentration of proteose peptone in the TPA production medium for the collagen gel culture was similar to that for the dish surface culture.     

Putative role of cholesteryl ester transfer protein in removal of cholesteryl ester from vascular interstitium, studied in a model system in cell culture

A model system to study the putative role of cholesteryl ester transfer protein in the egress of interstitial cholesteryl ester is described. Confluent cultures of bovine aortic smooth muscle cells were labeled for 24 h with [3H]cholesteryl linoleyl ether and [14C]cholesteryl linoleate by incubation with bovine milk lipoprotein lipase. This method of labeling results in the transfer of cholesteryl linoleyl ether and cholesteryl ester to three compartments: a trypsin-releasable, trypsin-resistant and catabolic compartment (Stein, O., Halperin, G., Leitersdorf, E., Olivecrona, T. and Stein, Y. (1984) Biochim. Biophys. Acta 795, 47-59). The efflux of labeled cholesteryl linoleyl ether and cholesteryl ester from the extracellular and cell-surface related compartments into a serum-free culture medium containing 1% bovine serum albumin was studied during 24 h of postincubation. The efflux was expressed as a percentage of pulse value, i.e., radioactivity retained by the cell culture at the end of the labeling period. The efflux of [3H]cholesteryl linoleyl ether, [14C]cholesteryl ester and 14C-labeled free cholesterol (formed by cellular hydrolysis of cholesterol ester) into the culture medium with 1% bovine serum albumin was about 5% of the pulse value. Addition of human lipoprotein-deficient serum resulted in a 3-10-fold increase in the efflux of [3H]cholesteryl linoleyl ether and [14C]cholesteryl ester, but did not change markedly the efflux of 14C-labeled free cholesterol. Rat lipoprotein-deficient serum which does not contain cholesteryl ester transfer protein did not increase the efflux of [3H]cholesteryl linoleyl ether or [14C]cholesteryl ester. The rate of cholesteryl ester efflux in the presence of human lipoprotein-deficient serum was linear for about 6 h and increased further up to 24 h. Addition of Intralipid to medium containing human lipoprotein-deficient serum further enhanced the efflux of [3H]cholesteryl linoleyl ether and, to a lesser extent, that of cholesteryl ester. A similar effect was observed also by addition of rat VLDL to medium containing human lipoprotein-deficient serum. Inhibition of cholesteryl linoleyl ether and cholesteryl ester efflux and marked enhancement of free cholesterol efflux occurred when rat HDL was added to medium containing human lipoprotein-deficient serum, while human HDL was only slightly inhibitory. The results obtained with human lipoprotein-deficient serum were reproduced with partially purified cholesteryl ester transfer protein. Using the partially purified cholesteryl ester transfer protein, the efflux of cholesteryl linoleate was compared to that of cholesteryl oleate and was found to be the same.