Effectiveness of autologous serum as an alternative to fetal bovine serum in adipose-derived stem cell engineering

In cell culture, medium supplemented with fetal bovine serum is commonly used, and it is widely known that fetal bovine serum supplies an adequate environment for culture and differentiation of stem cells. Nevertheless, the use of xenogeneic serum can cause several problems. We compared the effects of four different concentrations of autologous serum (1, 2, 5, and 10 %) on expansion and adipogenic differentiation of adipose-derived stem cells using 10 % fetal bovine serum as a control. The stem cells were grafted on nude mice and the in vivo differentiation capacity was evaluated. The isolation of adipose-derived stem cells was successful irrespective of the culture medium. The proliferation potential was statistically significant at passage 2, as follows: 10 % autologous serum >10 % fetal bovine serum = 5 % autologous serum >2 % autologous serum = 1 % autologous serum. The differentiation capacity appeared statistically significant at passage 4, as follows: 10 % fetal bovine serum >10 % autologous serum = 5 % autologous serum >2 % autologous serum = 1 % autologous serum. Ten percent autologous serum and 10 % fetal bovine serum had greater differentiation capacity than 1 and 2 % autologous serum in vivo, and no significant difference was observed between the groups at ≥5 % concentration at 14 weeks. In conclusion, 10 % autologous serum was at least as effective as 10 % fetal bovine serum with respect to the number of adipose-derived stem cells at the end of both isolation and expansion, whereas 1 and 2 % autologous serum was inferior.     

Optimization of Pre-transplantation Conditions to Enhance the Efficacy of Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) are considered a potential tool for cell based regenerative therapy due to their immunomodulatory property, differentiation potentials, trophic activity as well as large donor pool. Poor engraftment and short term survival of transplanted MSCs are recognized as major limitations which were linked to early cellular ageing, loss of chemokine markers during ex vivo expansion, and hyper-immunogenicity to xeno-contaminated MSCs. These problems can be minimized by ex vivo expansion of MSCs in hypoxic culture condition using well defined or xeno-free media i.e., media supplemented with growth factors, human serum or platelet lysate. In addition to ex vivo expansion in hypoxic culture condition using well defined media, this review article describes the potentials of transient adaptation of expanded MSCs in autologous serum supplemented medium prior to transplantation for long term regenerative benefits. Such transient adaptation in autologous serum supplemented medium may help to increase chemokine receptor expression and tissue specific differentiation of ex vivo expanded MSCs, thus would provide long term regenerative benefits.     

Evolution of the lymphoid system. I. Evidence for lymphocyte heterogeneity in rainbow trout revealed by the organ distribution of mitogenic responses.

Leukocytes from the various lymphoid tissues of rainbow trout (RBT) were tested for their capacity to respond to the lymphocyte mitogens concanavalin A (Con A), lipopolysaccharide (LPS), and purified protein derivative of tuberculin (PPD). Thymocytes responded to Con A but not to LPS or PPD. In contrast, leukocytes from anterior kidney were stimulated with LPS but not with Con A or PPD. Cells from spleen and peripheral blood were stimulated by each mitogen. However, the degree of stimulation at optimally stimulatory concentrations of each mitogen was distinctive. The finding that the patterns of mitogenic responses of cells from each tissue were significantly different suggested that there is lymphoid heterogeneity in the RBT with a unique tissue distribution. The species source of serum utilized as a medium supplement appeared to be capable of markedly affecting mitogenesis. Thus, LPS and PPD stimulation occurred in medium supplemented with rainbow trout serum (RBTS). On the other hand, LPS and PPD stimulation was not observed in medium supplemented with fetal bovine serum (FBS), with the exception of peripheral blood leukocytes which were stimulated by LPS in culture medium supplemented with FBS. Con A stimulated leukocytes from each lymphoid tissue in medium supplemented with RBTS and, with the exception of cells from anterior kidney, also stimulated cells from each tissue in medium supplemented with FBS. The kinetic profiles of the responses of peripheral blood leukocytes to Con A, LPS, and PPD suggested that the extent as well as the time required for maximal stimulation was dependent on the dose of mitogen.     

Reduced frequency of sister chromatid exchanges in human lymphocytes cultured with autologous serum

Summary The incidence of sister chromatid exchange (SCE) was determined in human lymphocytes cultured with fetal calf, human AB, and autologous serum. In each individual studied, cells grown in medium supplemented with fetal calf and human AB serums showed higher yields of SCE than those cultured with autologous serum. Increased concentration of fetal calf and human AB serum in the tissue culture medium resulted in elevated frequency of SCE. No such elevation in SCE frequency was observed with increased concentration of autologous serum. The results indicate the presence of extraneous SCE-inducing factors in fetal calf and human AB serum, the nature of which is not precisely known.Aided by C.S.I.R. Grant No. 7/45 (1052/77) EMR I     

Formation of bridges and large cellular clumps in CHO-cell microcarrier cultures: Effects of agitation dimethyl sulfoxide and calf serum

We have investigated conditions that inhibit the tendency of CHO K1 cells to form cellular bridges between microcarriers and dense clumps of cellular overgrowth in microcarrier cultures. Microcarrier aggregation by cellular bridge formation was found to occur only during periods of rapid cell growth. The level of microcarrier aggregation decreased with increasing agitation intensity. Dense masses of cellular overgrowth formed inside bridges connecting the microcarriers and in clumps that protruded off the microcarrier surface. To replace cells that were continuously sheared from the microcarriers, cell growth occurred preferentially in areas of overgrowth after confluent microcarriers were maintained in a serum-free medium. This ultimately led to poor surface coverage as bare spots developed on the microcarrier away from the areas of dense cellular overgrowth. The development of bare spots was inhibited when confluent microcarriers were maintained in medium supplemented with 1% serum. The development of cellular overgrowth was inhibited by dimethyl sulfoxide. Thus, maintaining confluent microcarriers in medium supplemented with 1% dimethyl sulfoxide and 1% calf serum resulted in microcarriers that appeared similar to monolayer cultures. There was also a decrease in bridging in cultures supplemented with either 1% calf serum or 1% dimethyl sulfoxide/1% calf serum compared to serum-free cultures.     

Differential recognition of tumor-derived and in vitro Epstein-Barr virus-transformed B-cell lines by fetal calf serum-specific T4-positive cytotoxic T-lymphocyte clones

Two interleukin-2 (IL-2)-dependent cytotoxic T-cell clones were obtained by limiting dilution from a lymphocyte culture stimulated in vitro with the autologous Epstein-Barr virus-transformed lymphoblastoid cell line (LCL) in the presence of fetal calf serum (FCS). Both clones uniformly had a T3+, T4+, Dr+ phenotype and lysed autologous B blasts, the autologous LCL, and allogeneic B cell lines sharing major histocompatibility complex (MHC) class II antigens. The cytotoxic function was triggered by FCS-derived components. There was no killing if the sensitive targets were cultured in serum-free medium or in medium supplemented with human serum. Sensitivity to lysis could be restored by exposing the targets to FCS for at least 6 hr at 37 degrees C. Monoclonal antibodies directed to T-cell-specific surface antigens and MHC class II antigens inhibited lysis with different efficiencies depending on the target cell origin. Killing of Burkitt's lymphoma (BL)-derived cell lines was blocked more easily than killing of LCLs. LCLs but not BL lines induced proliferation of the T-cell clones in the absence of exogenous IL-2. The differences were not related to quantitative variations in the expression of MHC class II antigens, indicating that BL lines differ from LCLs in other cell membrane properties that may influence antigen presentation. The results suggest that the affinity of effector/target binding, which is probably influenced by the concentration of antigenic determinants expressed on the target cell membrane, determines whether proliferative responses or cytotoxicity are induced in the antigen-recognizing T cells.     

Routine growth of cell lines in medium supplemented with milk instead of serum

Several cell lines can be grown in media, supplemented with milk instead of serum. To obtain a good plating efficiency the medium containing milk must be supplemented with calf serum at a final concentration of 0.5%. The milk must be centrifuged before use to get rid of the fat and the cellular debris. Using these precautions several monolayer cell lines can be grown routinely in milk supplemented media, just as well as in serum supplemented media. The economic advantages of using milk instead of serum are considerable.     

Long-term serum/plasma-free culture of human cytotoxic T lymphocytes induced from peripheral blood mononuclear cells

Tumor-specific human cytotoxic T lymphocytes (CTL) were induced by co-culturing peripheral blood mononuclear cells with X-ray-irradiated human lung squamous carcinoma cells, SQ-5, in the medium supplemented with interleukin(IL)-1, IL-2, IL-4 and IL-6, and 5% autologous plasma for 3 or 5 days. The CTL grew in serum/plasma-free medium containing these four interleukins and 0.5% bovine serum albumin for over a month and maintained kiling activity of target cells within 48 h at an effector/target ratio of 1.25. Their growth was essentially dependent on the target SQ-5 cells, which were renewed every 5 days. Under these conditions, IL-4 and IL-6 could be omitted. When anti-CD3 monoclonal antibody was added to the serum/plasma-free medium supplemented with IL-1 and IL-2, the target tumor cells were not required to maintain the specific killing activity of the CTL. A large number of CTL (10¹¹) were obtained in 35 days.     

Effective Elimination of Fibroblasts in Cultures of Melanocytes by Lowering Calcium Concentration in TPA Depleted Medium Following Geneticin Treatment

Melanocytes grow well in culture media supplemented with phorbol ester. However, phorbol ester is metabolically stable and has prolonged effects on multiple cellular responses. Different groups have reported that they could cultivate melanocytes without phorbol ester. However, in medium without phorbol esters contaminating fibroblasts are often difficult to eliminate. We have developed an improved method for eliminating fibroblasts using geneticin in serum free medium with bFGF, endothelin-1 and no phorbol ester. Treatment with a low concentration of geneticin effectively eliminated contaminating fibroblasts and was not harmful to melanocytes if geneticin was added initially in a high calcium media (2 mM) followed by a change to a low (0.09 mM) calcium media immediately after removal of the geneticin. With this technique we could easily establish pure melanocyte culture. This culture method will provide a useful tool for studies on melanocyte cellular response without the influence of phorbol ester.     

Improved Chemically Defined Basal Medium (CMRL-1969) for Primary Monkey Kidney and Human Diploid Cells

An improved tissue culture basal medium, CMRL-1969, supplemented with serum, has been evaluated by measuring the growth responses of primary cultures of trypsin-dispersed monkey kidney cells (PMKC) and of an established culture of a human diploid cell strain (HDCS). Medium H597, an early modification of medium 199 which has been used successfully in the preparation of poliomyelitis vaccine for 15 years, was used for comparison. In addition, parallel testing was done with Basal Medium Eagle (BME) widely used for the growth of HDCS. The improvements in basal medium CMRL-1969 are attributed to changes in amino acid concentrations, in vitamin composition, and, in particular, to enhanced buffering capacity. The latter has been achieved by the use of free-base amino acids and by increasing the dibasic sodium phosphate. The new medium has already been used to advantage for the production of polioviruses in PMKC where equivalent titers were obtained from cultures initiated with 70% of the number of cells required with earlier media. The population-doubling time was reduced in this system. Also, with small inocula of HDCS, the time required to obtain maximum cell yield was shorter with CMRL-1969 than with BME. Both media were supplemented with 10% calf serum. Maximum cell yields after repeated subcultivation in the new basal medium were greatly increased and the stability of the strain, as shown by chromosomal analysis, was not affected. Basal medium CMRL-1969 can be prepared easily in liquid or powdered form.     

Simultaneous identification of T and B cells in blood smears using antibody coated bacteria.

Rabbit lymph nodes produce in vitro more antibodies over longer periods if the culture medium is supplemented with a mixture of horse and fetal bovine serum rather than rabbit serum. Medium RPMI 1640 sustains longer antibody responses and promotes a greater expansion of the cultures than medium 199, both media being supplemented with the serum mixture. Propagation of fast-growing, antibody-producing cultures fed with RPMI 1640 was accomplished by interculture transfer of lymphoid tissue. Round, mostly nonadherent cells grow in RPMI 1640 and medium 199 but in the former a striking proliferation of fibroblast-like cells takes place. This proliferation does not occur in medium 199. Preliminary results indicate that although the fibroblast-like cells do not produce antibodies, they contribute to the response in vitro.     

Maintenance of bile acid synthesis and cholesterol 7 alpha-hydroxylase activity in cultured rat hepatocytes.

Addition of foetal-bovine serum to rat hepatocytes cultured in Williams E medium resulted in improved maintenance of bile-acid-synthetic capacity and cholesterol 7 alpha-hydroxylase activity as compared with cultures supplemented with rat or newborn-bovine serum or cultures in a hormonally defined serum-free medium. Minimally, 5% (v/v) foetal-bovine serum was necessary to maintain these liver-specific functions. Serum factor(s) responsible for these effects were not dialysable or associated with lipoproteins, but were removed by charcoal extraction.     

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.     

Effect of fractionation and rate of radiation dose on human leukemic cells, HL-60

The capacity of HL-60 cells, human acute promyelocytic leukemic cells established in culture, to repair sublethal radiation damage was estimated from the response of the cells to fractionated irradiation or to a single irradiation at different dose rates. The HL-60 cells grown as a suspension culture in RPMI 1640 medium supplemented with 10% calf serum and antibiotics showed a cloning efficiency of about 0.46 in an agar culture bed. After exposure of cells to a single dose of X rays at a dose rate of 78 rad/min, the survival curve was characterized by n = 2.5, Dq = 80 rad, and D0 = 83.2 rad. Split-dose studies demonstrated that the cells were able to repair a substantial portion of sublethal radiation damage in 2 hr. The response of the cells to irradiation at different dose rates decreased with a decrease in the dose rates, which could be attributed to repair of sublethal radiation damage. The radiation response of leukemic cells is only one of the many factors which affect the clinical outcome of total-body irradiation (TBI) followed by bone marrow transplantation. Nevertheless, the possibility that some of the malignant hemopoietic cells, if not all, may possess a substantial capacity to repair sublethal radiation damage should not be underestimated in planning total-body irradiation followed by bone marrow transplantation.     

Announcements

Human peripheral blood monocytes were reproducibly shown to lyse a variety of tumor cells in a 3- to 4-hr ⁵¹Cr release assay. Ficoll-Hypaque-purified mononuclear cells were suspended in medium supplemented with either 10% autologous serum or fetal calf serum (PCS). With either serum, highly purified (97–99%) and viable (>99%) monocyte suspensions were obtained by EDTA-reversible adherence to plastic surfaces which had been precoated with autologous serum. When used as effectors in cytotoxicity assays, the monocytes recovered from mononuclear cells suspended in FCS-supplemented medium exhibited higher cytolytic activity and were therefore used for further studies. Using FCS for both coating the plates and supplementing the suspension medium resulted in monocytes with low cytolytic activity. Tumor cell lysis measured by ⁵¹Cr release was detected within 2 hr of incubation and increased gradually with time. The level of lysis was dependent on the effector/target ratio and the tumor target cell employed. The involvement of natural killer lymphocytes in the observed tumoricidal activity was excluded. Detection of cytotoxic activity in a short-term assay will be very helpful in further studies of the mechanism of tumor cell killing by human monocytes since potential complicating effects of long-term in vitro cultivation will be minimized.     

Effects of FGF2 and FGF9 on osteogenic differentiation of bone marrow-derived progenitors

Bone repair is a major concern in reconstructive surgery. Transplants containing osteogenically committed mesenchymal stem cells (MSCs) provide an alternative source to the currently used autologous bone transplants which have limited supply and require additional surgery to the patient. A major drawback, however is the lack of a critical mass of cells needed for successful transplantation. The purpose of the present study was to test the effects of FGF2 and FGF9 on expansion and differentiation of MSCs in order to establish an optimal culture protocol resulting in sufficient committed osteogenic cells required for successful in vivo transplantation. Bone marrow-derived MSCs cultured in αMEM medium supplemented with osteogenic supplements for up to three passages (control medium), were additionally treated with FGF2 and FGF9 in various combinations. Cultures were evaluated for viability, calcium deposition and in vivo osteogenic capacity by testing subcutaneous transplants in nude mice. FGF2 had a positive effect on the proliferative capacity of cultured MSCs compared to FGF9 and control medium treated cultures. Cultures treated with FGF2 followed by FGF9 showed an increased amount of extracted Alizarin red indicating greater osteogenic differentiation. Moreover, the osteogenic capacity of cultured cells transplanted in immunodeficient mice revealed that cells that were subjected to treatment with FGF2 in the first two passages and subsequently to FGF9 in the last passage only, were more successful in forming new bone. It is concluded that the protocol using FGF2 prior to FGF9 is beneficial to cell expansion and commitment, resulting in higher in vivo bone formation for successful bone tissue engineering.     

Effect of serum-free and serum-containing medium on cellular levels of ER-based proteins in various mouse hybridoma cell lines

BiP, GRP94 and PDI, three endoplasmic reticulum (ER) based proteins are involved in the maturation of secretory proteins and might represent a bottleneck in the secretory pathway of monoclonal antibodies (MAB). With the three hybridoma cell lines tested, MAB production kinetics were significantly increased for the batch cultures done in serum-free medium (SFM) with respect to those done in serum-containing medium (SCM). It could be established that there was a correlation between the cellular levels of PDI and GRP94 and the specific MAB production rate. With respect to BiP, no correlation with the MAB production rate was observed. The non-producing myeloma cell line X63, used as a reference, showed increased cellular PDI levels when cultivated in SFM. However, in this cell, the cellular GRP94 levels were not significantly influenced by the medium composition.It was concluded that SFM induced an increase of cellular PDI levels and this elevation seemed to be responsible for the increase in the specific MAB production rates. On the other hand, only MAB producing cells showed an increase in the cellular GRP94 levels which might be a result of increased MAB sythesis. Indeed, I.13.17 cultivated in SFM supplemented with serum showed a significantly reduced (about 50%) specific MAB production rate in comparison to I.13.17 cultivated in non-serum supplemented SFM. The cellular PDI and BiP levels did not vary between these conditions of culture, whereas the cellular GRP94 level was about two-fold lower in I.13.17 cultivated in SFM when supplemented with serum than in I.13.17 cultivated in SFM without futher supplementation. These results are discussed with respect to the medium composition as well as in the context of apparent and potential bottlenecks within the secretory pathway of MAB. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 54: 165-180, 1997.     

Muscular differentiation of chicken myotubes in a simple defined synthetic culture medium and in serum supplemented media: Expression of the molecular forms of acetylcholinesterase

We attempted to cultivate muscle cells from chick embryos in a serum-free, defined medium similar to that proposed by Bottenstein and Sato (1979) for the growth and differentiation of a murine neuronal cell-line. (1) We found that muscle cells from the legs of 11-day old chick embryos can be cultivated in a medium containing the different components indicated by Bottenstein and Sato, with 2 g/l bovine serum albumin, without serum or chick embryo extract. Myoblasts attached to the gelatin-coated dishes without any addition of attachment factors. They differentiated into myotubes in a similar manner as in classical serum supplemented media. (2) The level of cellular AchE activity was comparable in cultures grown in the presence of fetal calf serum (FCS), of horse serum (HS) and in the defined medium. The percentage of A₁₂ form was however higher in the defined medium (25–30%) than in FCS supplemented medium (about 5–6%). In HS supplemented medium the A₁₂ form was not detectable, partly because horse serum contains immunoglobulins which bind chicken AChE. The addition of defined medium components to FCS medium cultures did not lead to an increase of A₁₂. In contrast, the addition of a small amount (1%) of fetal calf serum to DM cultures reduced the level of A₁₂ in a drastic manner. FCS components therefore seem to repress the biosynthesis of A₁₂ AChE, or increase its degradation. (3) We estimated intracellular and extracellular compartments of AChE. The ratio of endocellular to ectocellular AChE decreased with the age of the cultures. The G₁ form was intracellular at all stages analyzed, but the other molecular forms were located in both cellular compartment, in different proportion: A₁₂ and G₄ seemed to be located preferentially in the external compartment, whereas G₂ was preferentially intracellular. (4) Muscle cultures grown in the defined medium and in the presence of serum secreted globular forms of AChE in a similar manner.     

Effect of epidermal growth factor (EGF) and insulin on the kinetics of radiation-induced DNA lesions repair

To study the effect of both epidermal growth factor and insulin in terms of possible regulation of the repair process in cells, the time-course dependence of SSB and DSB repair have been investigated either in the presence of EGF (10 micrograms/ml) and insulin (1 microgram/ml) or without these factors in the medium (either supplemented with 10% serum or without serum) on Swiss 3T6 cells, exposed to ionizing radiation at a dose of 5-10 Gy using methods of neutral and alkaline elution as well as centrifugation on alkaline sucrose gradients. The absence of serum in the incubation medium during 18 to 24 hours before irradiation resulted in a sharp decrease in the rate of the repair of both single-strand break (SSB) and double-strand break (DSB). When cells were exposed to EGF and insulin immediately before irradiation the processes were restored to a significant extent. Data suggest that in the absence of other serum components, EGF and with insulin, are involved in the regulation of the repair of radiation-induced DNA lesions.