Serum-supplemented Culture Conditions Research Articles

Cultivation of an immortalized human corneal endothelial cell population and two distinct clonal subpopulations on thermo-responsive carriers

Recently, it was possible to show that human corneal endothelial cells (HCEC) can be cultured on thermo-responsive polymer substrates, and can be harvested as entire cell sheets without losing viability. We sought to study HCEC sheet cultivation on such cell culture carriers under serum-free conditions as the next consequential step in developing methods for generation of corneal endothelial cell transplants. An immortalized heterogenous HCEC population and two immortalized, clonally grown HCEC lines (HCEC-B4G12 and HCEC-H9C1) were cultured on thermo-responsive substrates under serum-supplemented and serum-free culture conditions. Cell sheets were characterized by phase contrast microscopy and by immunofluorescent staining for ZO-1, Na(+),K(+)-ATPase, and vinculin. All tested HCEC populations were able to adhere, spread and proliferate on thermo-responsive substrates under serum-supplemented conditions. Under serum-free conditions, pre-coating of the polymer substrates with ECM proteins was necessary to facilitate attachment and spreading of the cells, except in the case of HCEC-B4G12 cells. The heterogenous HCEC population formed closed monolayers, properly localized ZO-1 to lateral cell borders, and had moderate vinculin levels under serum-free, and higher vinculin levels under serum-supplemented culture conditions. HCEC-B4G12 cells formed closed monolayers, showed proper localization of ZO-1 and Na(+),K(+)-ATPase to lateral cell borders, and had high vinculin levels irrespective of culture conditions. In contrast, HCEC-H9C1 cells had lowest vinculin levels under serum-supplemented, and higher vinculin levels under serum-free culture conditions. ZO-1 was detected throughout the cytoplasm under both culture conditions. These loosely adherent cells were only able to form a closed monolayer under serum-supplemented conditions. Serum-free production of HCEC sheets is possible. The extremely adherent clonal HCEC line B4G12 produced higher vinculin levels than the other two tested HCEC populations, and showed strong adherence to the thermo-responsive, polymeric culture substratum irrespective of culture conditions. This cell line closely resembles terminally differentiated HCEC in vivo, and was found to be particularly suitable for further studies on HCEC cell sheet engineering.

Estrogen mitogenic action. I. Demonstration of estrogen-dependent MTW9/PL2 carcinogen-induced rat mammary tumor cell growth in serum-supplemented culture and technical implications.

The MTW9/PL cell line was established by our laboratory in culture from the carcinogen-induced hormone-responsive MT-W9A rat mammary tumor of a Wistar-Furth (W/Fu) rat. This tumor formed estrogen, androgen, and progesterone responsive tumors in W/Fu rats (Sirbasku, D. A., Cancer Res. 38:1154-1165; 1978). It was later used to derive the MTW9/PL2 cell population which was also estrogen-responsive in vivo (Danielpour, D., et al., In Vitro Cell. Dev. Biol. 24:42-52; 1988). In the study presented here, we describe serum-supplemented culture conditions in which the MTW9/PL2 cells demonstrate > or = 80-fold steroid hormone growth responses. All sera used were steroid hormone-depleted by charcoal-dextran treatment at 34 degrees C. The studies were done with horse serum as well as serum from other mammalian species. The growth of the MTW9/PL2 cells was biphasic in response to hormone-depleted serum. Concentrations of < or = 5% (v/v) promoted optimum growth. Above this concentration, serum was inhibitory. Concentrations > or = 40% (v/v) inhibited growth altogether. Addition of 1.0 x 10(-13)-1.0 x 10(-8) M 17beta,-estradiol (E2) reversed the inhibition completely. At 1.0 x 10(-8) M, estrone, estriol and diethylstilbestrol promoted growth as well as E2. Testosterone and dihydrotestosterone promoted growth only at > or = 10(-7) M. Progesterone was effective only at > or = 10(-6) M. Cortisol was ineffective. Labeled-hormone-binding analysis and Western immunoblotting documented that MTW9/PL2 cells had estrogen and progesterone receptors but not androgen or cortisol receptors. Estrogen treatment of MTW9/PL2 cells induced a concentration and time dependent increase in progesterone receptors. We conclude (1) the MTW9/PL2 population is the first highly steroid hormone-responsive rat mammary tumor cell line to be established in culture from a carcinogen-induced tumor, and (2) sera from a number of species including horse, rat and human contain an inhibitor which mediates estrogen sensitive MTW9/PL2 cell growth in culture.

Signaling through interleukin-6 receptor supports blast cell proliferation in acute myeloblastic leukemia.

The role of the interleukin-6/interleukin-6 receptor (IL-6/IL-6R) system in regulating blast cell growth in 8 acute myeloblastic leukemia (AML) patient-derived cell lines was investigated. As they all expressed IL-6R and as none of them responded to exogenous IL-6 under conventional serum-supplemented culture conditions, we investigated whether signaling through IL-6R plays any role in maintaining their spontaneous colony growth. This was done by treating the cells with monoclonal antibodies made against the ligand-specific IL-6R alpha-chain or the signal transducer gp130. In serum-supplemented cultures inhibition of gp130 function did not affect the cell line growth, whereas anti-IL-6R alpha-chain antibody reduced colony growth. While some of the cell lines also showed similar growth characteristics in a serum-free environment, some others changed their growth pattern and stopped responding to anti-IL-6R alpha-chain treatment. At the same time, these cell lines also began to respond to exogenously added IL-6 and, interestingly, were stimulated by anti-gp130 antibody. Hence, in some of the blast cells, clonogenic cell growth seemed to be also negatively controlled by an endogenously produced growth-depressing cytokine or cytokines that utilize gp130. All the cell lines, whether cultured in the presence or absence of serum expressed IL-6 both at mRNA and protein level. The current results indicate that AML cells can use IL-6 as a growth stimulating factor, supplied either paracrinely or autocrinely. This could implicate the use of anti-IL-6R alpha-chain antagonists in AML treatment, not IL-6.

Further characterization of a new in vitro angiogenesis model under serum free culture conditions; suppression of endothelial cell differentiation by serum

We studied the regulation of the extracellular matrix in the recently established murine vascular endothelial cell clones, F-2 or F-2C. F-2 cells constitutively show a cobblestone growth pattern under serum supplemented culture conditions, whereas F-2C cells undergo spontaneous histodifferentiation to form tubular structures in chemically defined media. We reported that the tubulogenesis induced by F-2C might relate to the heavy deposition of a `basement membrane analog' as a subendothelial matrix (SEM). We further characterized the regulation of extracellular matrix (ECM) metabolism in these cell clones, in terms of gelatinase expression, ECM degradation and the effects of serum. F-2C cells in culture medium containing 1% serum did not undergo tubulogenesis but presented cobblestone growth. Zymography analysis showed that both F-2 and F-2C cells express two gelatinases (72 and 92 kDa). However, F-2 cells mainly expressed the former and faintly the latter, whereas F-2C mainly expressed the latter. Proteolysis studies showed that the spent media conditioned by F-2C cells partially cleaved type IV collagen and completely degraded type V collagen. The cleavage of type V collagen was suppressed by the addition of serum, whereas that of type IV collagen was not. The proteolysis of laminin and fibronectin by the conditioned medium was not observed. Serum-supplemented F-2 or F-2C cultures markedly suppressed SEM deposition. These results indicated that F-2C cells under serum free culture conditions not only present a simple and useful in vitro model with which to study the dynamic processes of proteolysis and ECM metabolism during the sequential phases of angiogenesis, but is also useful for analyzing the serum effects on angiogenesis (AG).

Effects of vitamin D-binding proteins on HL-60 cell differentiation induced by 26,26,26,27,27,27-hexafluoro-1α, 25-dihydroxyvitamin D 3

Monocytic differentiation-inducing activity of 26,26,26,27,27,27-hexafluoro-1α,25-dihydroxyvitamin D 3 [26,27-F 6-1α,25-(OH) 2D 3] was re-evaluated in human promyelocytic leukemia (HL-60) cells in serum-supplemented or serum-free culture. The order of in vitro potency for reducing nitroblue tetrazolium (NBT) was 26,27-F 6-1 α,25-(OH) 2D 3 > 1 α,25-dihydroxyvitamin D 3 [1 α,25-(OH) 2D 3] = 26,26,26,27,27,27-F 6-1 α,23( S),25-trihydroxyvitamin D 3[26,27-F 6-1 α,23( S),25-(OH) 3D 3] under serum-supplemented culture conditions, whereas the order was 1 α, 25-(OH) 2D 3 = 26,27-F 6-1 α,25-(OH) 2D 3 > 26,27-F 6-1 α,23(S), 25-(OH) 3D 3 under serum-free culture conditions. This rank order for differentiation-inducing activity under serum-free culture conditions correlated well with the binding affinity of these analogs for vitamin D 3 receptor of HL-60 cells. The order of relative % binding affinity for the vitamin D-binding protein in fetal calf serum was 1 α,25-(OH) 2D 3 (100%)⪢26,27-F 6-1 α,25-(OH) 2D 3(5.1%) > 26,27-F 6-1 α,23( S),25-(OH) 3D 3 (<1%). These results suggest that serum vitamin D-binding proteins apparently modulate monocytic differentiation of HL-60 cells by 26,27-F 6- 1α,25-(OH) 2D 3 under serum-supplemented culture conditions.

Effects of novel 26,27-dialkyl analogs of 1α,25-dihydroxyvitamin D 3 on differentiation-inducing activity of human promyelocytic leukemia (HL-60) cells in serum-supplemented or serum-free culture

Monocylic differentiation-inducing activity of steroidal side chain-lengthened 26,27-dialkyl analogs of 1α,25-dihydroxyvitamin D 3 was examined in human promyelocytic leukemia (HL-60) cells in serumsupplemented or serum-free culture. The order of in vitro potency for reducing nitroblue tetrazolium was 1α,25-dihydroxy-26,27-dimethylvilamin D 3, ≧ 1α,25-dihydroxyvitamin D 3 > 1α,25-dihydroxy-26,27-diethylvitamin D 3 ⪢ 1 α,25-dihydroxy-26,27-dipropylvitamin D 3 under serum-free culture conditions. Analysis by sucrose density-gradient centrifugation or polyethylene glycol precipitation technique showed that the potency order for differentiation-inducing activity correlated well with binding affinity of these analogs for vitamin D 3; receptor of HL-60 cells. Under serum-supplemented culture conditions, the lack of correlation between biologic activity and analog-binding affinity for receptor was caused by differences in binding affinity of these analogs for serum vitamin D-binding proteins. These results suggest that serum vitamin D-binding proteins apparently modulate monocytic differentiation of HL-60 cells by these analogs under serum-supplemented culture conditions.

Effect of mitogens in various organs and cell culture conditioned media on rat oligodendrocytes.

Extracts prepared from embryonic, neonatal and adult rat brain were examined for the presence of oligodendroglial mitogens. Brain-derived mitogenic activities were found in all developmental stages with specific activity increasing during neonatal development. Extracts from postnatal day 7 contained the highest specific activity. Upon fractionation by molecular weight, each developmental stage expressed a peak of mitogenic activity corresponding to 67,000 daltons. This fraction was able to induce proliferation in cultures grown in serum, serumless chemically defined medium or serum-free medium alone. Neonatal and adult brain extracts had an additional peak of activity at 14,000 daltons. This latter activity was expressed only under serum-supplemented culture conditions. Mitogenic activity was also found in conditioned media from the clonal glioma cell line C6 and primary astrocytes and in extracts derived from neonatal rat liver. These data indicate that a limited range of brain-derived mitogens for oligodendrocytes exist during development and adulthood.

Serum-free medium for cultures of the postnatal mouse cerebellum: Only insulin is essential

Serum-free culture conditions for dissociated postnatal mouse cerebellar cells were investigated. This study demonstrates that among various supposed growth-promoting factors only insulin is required as an additive to the basic medium. If viewed by phase contrast microscopy, cultures kept in insulin-supplemented basic medium looked identical to those maintained in the presence of a mixture of growth-promoting factors (insulin, putrescine, transferrin, progesterone, triiodothyronine, selenium). In addition, quantitative evidence is provided indicating that cellular survival is supported to the same extent by insulin as by this admixture. Insulin concentrations required ranged between 0.3–20 μg/ml. By contrast to serum-supplemented culture conditions, no significant proliferation of non-neuronal cells was observed in serum-free culture media. It is expected that by these findings attention will be focused again to the exact role insulin plays in the central nervous system.

Replacement of serum by insulin, transferrin, albumin, phosphatidyl choline, cholesterol, and some trace elements in cultures of mouse myeloid leukemia cells sensitive to inducers of differentiation

In serum-supplemented culture conditions, mouse myeloid leukemia cells (Ml) could be induced by various inducers including dexamethasone to form Fc receptors, phagocytize, migrate in agar, induce lysosomal enzymes, and change into forms that were morphologically similar to macrophages and granulocytes. Ml cells grew in the absence of serum in synthetic medium supplemented with insulin, transferrin, and several trace elements. The cells were maintained for more than 6 months in this medium. Induction of differentiation by dexamethasone was obtained with the cells that had been grown in serum-free medium for several days. However, the ability to be induced to differentiate in this medium was then lost with further growth of the cells in this medium. These cells could be induced to differentiate by dexamethasone with the aid of albumin, phosphatidyl choline, and cholesterol, but not by the inducer alone. The cells were leukemogenic in syngeneic mice. Macrophage-like cells that developed spontaneously from Ml cells were continuously maintained in this environment without morphological alteration or loss of their differentiated properties such as formation of Fc receptors, phagocytosis, and lysozyme synthesis.