Keratinocytes Propagated in Serum-Free, Feeder-Free Culture Conditions Fail to Form Stratified Epidermis in a Reconstituted Skin Model

Dendritic cell (DC)-based antitumor vaccines have proven to be a safe approach, but often fail to generate robust results between trials. Translation to the clinic has been hindered in part by the lack of standard operation procedures for vaccines production, namely the definition of optimal culture conditions during ex-vivo DC differentiation. Here we sought to compare the ability of three clinical grade serum-free media, DendriMACS, AIM-V, and X-VIVO 15, alongside with fetal bovine serum-supplemented Roswell Park Memorial Institute Medium (RPMI), to support the differentiation of monocyte-derived DCs (Mo-DCs). Under these different culture conditions, phenotype, cell metabolomic profiles, response to maturation stimuli, cytokines production, allogenic T cell stimulatory capacity, as well as priming of antigen-specific CD8+ T cells and activation of autologous natural killer (NK) cells were analyzed. Immature Mo-DCs differentiated in AIM-V or X-VIVO 15 presented lower levels of CD1c, CD1a, and higher expression of CD11c, when compared to cells obtained with DendriMACS. Upon stimulation, only AIM-V or X-VIVO 15 DCs acquired a full mature phenotype, which supports their enhanced capacity to polarize T helper cell type 1 subset, to prime antigen-specific CD8+ T cells and to activate NK cells. CD8+ T cells and NK cells resulting from co-culture with AIM-V or X-VIVO 15 DCs also showed superior cytolytic activity. 1H nuclear magnetic resonance-based metabolomic analysis revealed that superior DC immunostimulatory capacities correlate with an enhanced catabolism of amino acids and glucose. Overall, our data highlight the impact of critically defining the culture medium used in the production of DCs for clinical application in cancer immunotherapy. Moreover, the manipulation of metabolic state during differentiation could be envisaged as a strategy to enhance desired cell characteristics.

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PRODUCTION OF HUMAN INTERLEUKIN-2(TCGF) USING SERUM FREE CULTURE CONDITIONS

Human Lymphokines ◽

10.1016/b978-0-12-406080-7.50017-5 ◽

1982 ◽

pp. 135-145

Author(s):

Laurence B. Sohook ◽

Flemming Kristensen ◽

Ursula Otz ◽

Sandor Lazary ◽

Alain L. de Weck

Keyword(s):

Interleukin 2 ◽

Culture Conditions ◽

Serum Free

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The developmental potential of mouse oocytes matured in serum-free culture conditions

Human Reproduction ◽

10.1093/oxfordjournals.humrep.a136181 ◽

1995 ◽

Vol 10 (7) ◽

pp. 1810-1815 ◽

Cited By ~ 3

Author(s):

Ricardo T. Serta ◽

Jiannis Michalopoulos ◽

Machelle M. Seibel ◽

Ann A. Kiessling

Keyword(s):

Culture Conditions ◽

Developmental Potential ◽

Serum Free ◽

Mouse Oocytes

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Proliferation and Survival of Mammary Carcinoma Cells Are Influenced by Culture Conditions Used for Ex Vivo Expansion of CD34+Blood Progenitor Cells

Blood ◽

10.1182/blood.v93.2.746 ◽

1999 ◽

Vol 93 (2) ◽

pp. 746-755 ◽

Cited By ~ 11

Author(s):

A. Spyridonidis ◽

W. Bernhardt ◽

D. Behringer ◽

G. Köhler ◽

M. Azemar ◽

...

Keyword(s):

Breast Cancer ◽

Tumor Cells ◽

Mammary Carcinoma ◽

Ex Vivo ◽

Culture Conditions ◽

Ex Vivo Expansion ◽

Carcinoma Cells ◽

Serum Free ◽

Cellular Contact ◽

Mammary Carcinoma Cells

Abstract Malignant cell contamination in autologous transplants is a potential origin of tumor relapse. Ex vivo expansion of CD34+ blood progenitor cells (BPC) has been proposed as a tool to eliminate tumor cells from autografts. To characterize the influence of culture conditions on survival, growth, and clonogenicity of malignant cells, we isolated primary mammary carcinoma cells from pleural effusions and ascites of patients with metastatic breast cancer and cultured them in the presence of stem cell factor (SCF), interleukin-1β (IL-1β), IL-3, IL-6, and erythropoietin (EPO), ie, conditions previously shown to allow efficient ex vivo expansion of CD34+ BPC. In the presence of serum, tumor cells proliferated during a 7-day culture period and no significant growth-modulatory effect was attributable to the presence of hematopoietic growth factors. When transforming growth factor-β1 (TGF-β1) was added to these cultures, proliferation of breast cancer cells was reduced. Expansion of clonogenic tumor cells was seen in the presence of SCF + IL-1β + IL-3 + IL-6 + EPO, but was suppressed by TGF-β1. Cocultures of tumor cells in direct cellular contact with hematopoietic cells showed that tumor cell growth could be stimulated by ex vivo expanded hematopoietic cells at high cell densities (5 × 105/mL). In contrast, culture under serum-free conditions resulted in death of greater than 90% of breast cancer cells within 7 days and a further decrease in tumor cell numbers thereafter. In the serum-free cultures, hematopoietic cytokines and cellular contact with CD34+ BPC could not protect the tumor cells from death. Therefore, ex vivo expansion of CD34+ BPC in serum-free medium provides an environment for efficient purging of contaminating mammary carcinoma cells. These results have clinical significance for future protocols in autologous progenitor cell transplantation in cancer patients.

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110. EFFECTS OF cAMP AND SERUM ON HUMAN TROPHOBLAST CELL VIABILITY AND DIFFERENTIATION

Reproduction Fertility and Development ◽

10.1071/srb09abs110 ◽

2009 ◽

Vol 21 (9) ◽

pp. 29

Author(s):

Y. Chen ◽

M. Allars ◽

C. Abou-Seif ◽

R. C. Nicholson

Keyword(s):

Cell Viability ◽

Syncytium Formation ◽

Culture Conditions ◽

Trophoblast Cell ◽

Bewo Cell ◽

Trophoblast Cells ◽

Human Trophoblast ◽

Serum Free ◽

Bewo Cells ◽

Free Media

The formation of syncytium is a pivotal event for trophoblast cells to interact with the placental bed. While cAMP is regarded as an inducer of syncytialisation, the affect of different culture conditions on this cAMP effect has not been explored. Therefore, the effects of cAMP on cell differentiation and viability in the presence or absence of serum were investigated in the human choriocarcinoma cell lines, BeWo and JEG-3. We observed that in the absence of cAMP, BeWo cells grew best in media containing 10% FCS, followed by media containing charcoal-stripped 10% FCS (10%CCS), and less well in serum-free media. In the presence of cAMP ( 0.25~1.5 mM ), our observations suggest different cellular programmes may be in play. Treatment of BeWo cells with 0.75 mM cAMP for 24h and 48h, in the absence of serum, increased cell viability (MTT assay) by 25.1% and 46.1% respectively, compared to the control cells. Interestingly, this cAMP effect on cell viability was not observed in the JEG-3 cell line. In contrast, BeWo cell viability was decreased by 49.5% and 25.2%, and by 27.5% and 31.1% in JEG-3 cells, when the cAMP stimulated cells were cultured for 48h in 10% CCS and 10% FCS media, respectively. In addition, we observed a change in BeWo, but not JEG-3, cell morphology to a spindle-like shape with pseudopodia when cAMP stimulated cells were cultured in media containing 10% CCS or 10% FCS for greater than 24h. Since the process of syncytialisation may involve apoptotic events, we speculate that the different effects of cAMP on cell viability in trophoblast cells may be related to syncytialising factors contained in serum media. Further study will clarify whether serum promotes syncytium formation, while the lack of serum based factors could switch the cellular programme from one of syncytialisation toward a more proliferative type.

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The Influence of Serum-Free Culture Conditions on Skeletal Muscle Differentiation in a Tissue-Engineered Model

Tissue Engineering ◽

10.1089/ten.2007.0095 ◽

2008 ◽

Vol 14 (1) ◽

pp. 161-171 ◽

Cited By ~ 1

Author(s):

Debby Gawlitta ◽

Kristel J.M. Boonen ◽

Cees W.J. Oomens ◽

Frank P.T. Baaijens ◽

Carlijn V.C. Bouten

Keyword(s):

Skeletal Muscle ◽

Muscle Differentiation ◽

Culture Conditions ◽

Skeletal Muscle Differentiation ◽

Serum Free

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MICROCARRIER CULTURE OF HUMAN ENDOTHELIAL CELL TYPES - A SOURCE OF METABOLITES

10.1055/s-0038-1643348 ◽

1987 ◽

Author(s):

N R Hunter ◽

I R MacGregor ◽

J Dawes ◽

D S Pepper

Keyword(s):

Endothelial Cell ◽

Cell Types ◽

Culture Conditions ◽

Human Endothelial Cell ◽

Cell Protein ◽

Microcarrier Culture ◽

Serum Free ◽

Cytodex 3 ◽

Free Media ◽

Microcarrier Cultures

The production of human endothelial cell secretory products in amounts sufficient for biochemical studies is largely restricted by the culture growth area. Conventional flat bed systems yield at best 20-30 x 106 cells per 180cm2 culture flask. To overcome this problem, cells may be grown on Cytodex 3 microcarriers allowing large numbers of cells to be grown and conditioned in small culture volumes. A typical microcarrier unit will contain 200-300 x 106 cells and may be expanded in excess of 1000 x 106 cells at confluence. High viability (95%) and recovery (70-80%) in sub-culturing of microcarrier to microcarrier culture can be achieved with careful management of culture conditions and brief exposure to enzymes.Human umbilical artery and vein, and saphenous vein endothelial cells were prepared and grogn on microcarrier cultures to cell populations of 200-450 x 106 cells and conditioned for 14 day periods in serum-free media.The production profiles of several endothelial cell proteins including thrombospondin (TSP), von Willebrand Factor (vWF) and issue plasminogen activator (t-PA) were measured by radioimmunoassay under these conditions, and demonstrate the use of microcarrier cultures in producing milligram quantities of engothelial cell protein. For example, a HUVEC culture of 200 x 106 cells conditioned with serum-free media for 14 days yielded a total of 6.9mg TSP, 0.7mg vWF and 48.9ug t-PA. In this laboratory one such application of the system was the purification of endothelial proteins in amounts sufficient for immunisation of mice prior to the production of monoclonal antibodies and for subsequent characterisation.

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