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1.
Ding Huang Wen-Juan Peng Qian Ye Xu-Ping Liu Liang Zhao Li Fan Kang Xia-Hou Han-Jing Jia Jian Luo Lin-Ting Zhou Bei-Bei Li Shi-Lei Wang Wen-Ting Xu Ze Chen Wen-Song Tan 《PloS one》2015,10(11)
Development of serum-free suspension cell culture processes is very important for influenza vaccine production. Previously, we developed a MDCK suspension cell line in a serum-free medium. In the present study, the growth kinetics of suspension MDCK cells and influenza virus production in the serum-free medium were investigated, in comparison with those of adherent MDCK cells in both serum-containing and serum-free medium. It was found that the serum-free medium supported the stable subculture and growth of both adherent and suspension cells. In batch culture, for both cell lines, the growth kinetics in the serum-free medium was comparable with those in the serum-containing medium and a commercialized serum-free medium. In the serum-free medium, peak viable cell density (VCD), haemagglutinin (HA) and median tissue culture infective dose (TCID50) titers of the two cell lines reached 4.51×106 cells/mL, 2.94Log10(HAU/50 μL) and 8.49Log10(virions/mL), and 5.97×106 cells/mL, 3.88Log10(HAU/50 μL), and 10.34Log10(virions/mL), respectively. While virus yield of adherent cells in the serum-free medium was similar to that in the serum-containing medium, suspension culture in the serum-free medium showed a higher virus yield than adherent cells in the serum-containing medium and suspension cells in the commercialized serum-free medium. However, the percentage of infectious viruses was lower for suspension culture in the serum-free medium. These results demonstrate the great potential of this suspension MDCK cell line in serum-free medium for influenza vaccine production and further improvements are warranted. 相似文献
2.
Zea Borok Spencer I. Danto Stephanie M. Zabski Edward D. Crandall 《In vitro cellular & developmental biology. Animal》1994,30(2):99-104
Summary Isolated type II pneumocytes grown in serum on tissue culture-treated polycarbonate filters form monolayers with characteristic
bioelectric properties, and change morphologically with time in culture to resemble type I cells. Concurrently, the cells
express type I cell surface epitopes, making this a potentially useful in vitro model with which to study regulation of alveolar
epithelial cell function and differentiation. To define specific soluble growth factors and matrix substances that may regulate
these processes, it would be preferable to culture isolated pneumocytes de novo under completely defined, serum-free conditions.
In this study, we developed a completely defined serum-free medium that is capable of supporting alveolar epithelial cells
in primary culture, allowing the formation of monolayers with characteristic bioelectric and phenotypic properties. Freshly
isolated rat type II cells were resuspended in completely defined serum-free medium and plated de novo on polycarbonate filters.
Plating efficiency, bioelectric properties, morphology, and binding of a type I cell-specific monoclonal antibody were determined
as functions of time. Plating efficiency plateaus at about 14% by Day 3 in culture. Transepithelial resistance rises to high
levels, peaking at 1.76±0.14 KΩ-cm2 by Day 5 in culture. Short-circuit current peaks on Day 3 in culture at 2.71±0.35 μA/cm2. With time, the cells gradually become flattened with protuberant nuclei and long cytoplasmic extensions, more closely resembling
type I cells, and begin to express a type I cell surface epitope. These observations indicate that it is feasible to culture
alveolar epithelial cell monolayers under completely defined serum-free conditions de novo. This culture system should prove
useful for identifying soluble growth factors and matrix substances that modulate alveolar epithelial cell biological properties. 相似文献
3.
ARPE-19 conditioned medium promotes neural differentiation of adipose-derived mesenchymal stem cells
Giuliana Mannino Martina Cristaldi Giovanni Giurdanella Rosario Emanuele Perrotta Debora Lo Furno Rosario Giuffrida Dario Rusciano 《World journal of stem cells》2021,13(11):1783-1796
BACKGROUNDAdipose-derived stem cells (ASCs) have been increasingly explored for cell-based medicine because of their numerous advantages in terms of easy availability, high proliferation rate, multipotent differentiation ability and low immunogenicity. In this respect, they have been widely investigated in the last two decades to develop therapeutic strategies for a variety of human pathologies including eye disease. In ocular diseases involving the retina, various cell types may be affected, such as Müller cells, astrocytes, photoreceptors and retinal pigment epithelium (RPE), which plays a fundamental role in the homeostasis of retinal tissue, by secreting a variety of growth factors that support retinal cells.AIMTo test ASC neural differentiation using conditioned medium (CM) from an RPE cell line (ARPE-19).METHODSASCs were isolated from adipose tissue, harvested from the subcutaneous region of healthy donors undergoing liposuction procedures. Four ASC culture conditions were investigated: ASCs cultured in basal Dulbecco''s Modified Eagle Medium (DMEM); ASCs cultured in serum-free DMEM; ASCs cultured in serum-free DMEM/F12; and ASCs cultured in a CM from ARPE-19, a spontaneously arising cell line with a normal karyotype derived from a human RPE. Cell proliferation rate and viability were assessed by crystal violet and MTT assays at 1, 4 and 8 d of culture. At the same time points, ASC neural differentiation was evaluated by immunocytochemistry and western blot analysis for typical neuronal and glial markers: Nestin, neuronal specific enolase (NSE), protein gene product (PGP) 9.5, and glial fibrillary acidic protein (GFAP).RESULTSDepending on the culture medium, ASC proliferation rate and viability showed some significant differences. Overall, less dense populations were observed in serum-free cultures, except for ASCs cultured in ARPE-19 serum-free CM. Moreover, a different cell morphology was seen in these cultures after 8 d of treatment, with more elongated cells, often showing cytoplasmic ramifications. Immunofluorescence results and western blot analysis were indicative of ASC neural differentiation. In fact, basal levels of neural markers detected under control conditions significantly increased when cells were cultured in ARPE-19 CM. Specifically, neural marker overexpression was more marked at 8 d. The most evident increase was observed for NSE and GFAP, a modest increase was observed for nestin, and less relevant changes were observed for PGP9.5. CONCLUSIONThe presence of growth factors produced by ARPE-19 cells in tissue culture induces ASCs to express neural differentiation markers typical of the neuronal and glial cells of the retina. 相似文献
4.
Yuhsi Matuo Nozomu Nishi Hideyuki Tanaka Ikuharu Sasaki John T. Issacs Fumio Wada 《In vitro cellular & developmental biology. Plant》1988,24(10):1053-1056
Summary AT-3 cells, one of anaplastic cell lines established from the Dunning prostatic carcinoma of rats, were able to grow under
serum-free conditions in a state of suspension detached from a substratum. Radioimmunoassays using monoclonal antibody against
rat insulin-like growth factor II (IGF-II) revealed the presence of IGF-II-related peptide in acid-ethanol extracts extracsts
of lyophilized serum-free media conditioned by AT-3 cell. The peptide contents in the culture media increased with increase
in cell number; 71 ng at 3.0 × 106 cells and 449 ng at 4.6 × 107 cells. IGF-II-related peptide was hardly detectable in acid-ethanol extracts of AT-3 cells harvested after 13-days culture.
These results indicate that AT-3 cells produce IGF-II-related peptide ana may release it into the culture media.
Editor's statement One or more members of the insulin-like growth factor family have been established previously as mitogen
for isolated prostate cells. This report suggests that IGF-II member of the family may be involved in autocrine support of
cells from highly malignant prostate tumors. 相似文献
5.
Egorova-Zachernyuk TA Bosman GJ Degrip WJ 《Applied microbiology and biotechnology》2011,89(2):397-406
Uniform stable-isotope labeling of mammalian cells is achieved via a novel formulation of a serum-free cell culture medium
that is based on stable-isotope-labeled autolysates and lipid extracts of various microbiological origin. Yeast autolysates
allow complete replacement of individual amino acids and organic acids in a chemically defined medium (DMEM/F12), enabling
a cost-effective formulation of a stable-isotope-labeled culture medium for mammalian cells. In addition, biomass-derived
hydrolysates, autolysates, and lipid extracts of various classes of algae were explored as cell culture components, both separately
and in combination with yeast autolysates. Optimal autolysate concentrations were established. Such novel medium formulations
were tested on mammalian cell lines, often used for recombinant protein production, i.e., Chinese hamster ovary (CHO) and
human embryonic kidney (HEK 293). Special attention was paid to the adaptation of these mammalian cell lines to serum-free
media. Formulation of the novel proprietary cell culture medium PLIm, based on yeastolates instead of individual amino acids
and organic acids, allows a four- to eightfold cost reduction for 15N and 13C,15N stable-isotope-labeling, respectively, in CHO cells and a three- to sixfold cost reduction in HEK 293 cells. A high level
of stable-isotope enrichment of mammalian cells (>90%) was achieved within four passages by complete replacement of carbon
and nitrogen sources in the medium with their stable-isotope-labeled analogs. These conditions can be used to more cost-effectively
produce labeled recombinant proteins in mammalian cells. 相似文献
6.
《Journal of Fermentation Technology》1986,64(2):145-153
A clone, AH-01S, derived from a human monocytic leukemia cell line, THP-1, grew rapidly in a serum-free medium containing insulin, transferrin, ethanolamine, and sodium selenite. In batch culture using the serum-free medium, the AH-01S cells proliferated at a specific growth rate (μ) of 0.30 to 0.50 (1/day) from a cell concentration of 1 × 104 cells/ml to 1.6 × 106 cells/ml, an increase of 160 times. A higher cell concentration of 0.45 × 107 cells/ml (cell volume ratio was 0.5%) was obtained in spinner flask culture using the serum-free medium. A mean specific growth rate 0.50 (1/day) was also observed in a culture in a fully instrumented cell culture fermentor. However, μ decreased drastically after the cell concentration reached 1.5 × 106 cells/ml. Analyses of medium composition during cultivation revealed that under lower cell concentration, l-glutamine was the main carbon source while glucose was converted to lactate almost stoichiometrically, and that the production of lactate from glucose decreased at higher cell concentrations. To obtain cultures of 1 × 109 cells, 1,200 to 1,300 mg of a carbon source (glucose) and 400 to 500 of amino acids were consumed during high cell concentration cultivation of the AH-01S cells in the serum-free medium. 相似文献
7.
The primary culture of neuronal cells plays an important role in neuroscience. There has long been a need for methods enabling the long-term culture of primary neurons at low density, in defined serum-free medium. However, the lower the cell density, the more difficult it is to maintain the cells in culture. Therefore, we aimed to develop a method for long-term culture of neurons at low density, in serum-free medium, without the need for a glial feeder layer. Here, we describe the work leading to our determination of a protocol for long-term (>2 months) primary culture of rat hippocampal neurons in serum-free medium at the low density of 3×104 cells/mL (8.9×103 cells/cm2) without a glial feeder layer. Neurons were cultured on a three-dimensional nanofibrous hydrogel, PuraMatrix, and sandwiched under a coverslip to reproduce the in
vivo environment, including the three-dimensional extracellular matrix, low-oxygen conditions, and exposure to concentrated paracrine factors. We examined the effects of varying PuraMatrix concentrations, the timing and presence or absence of a coverslip, the timing of neuronal isolation from embryos, cell density at plating, medium components, and changing the medium or not on parameters such as developmental pattern, cell viability, neuronal ratio, and neurite length. Using our method of combining the sandwich culture technique with PuraMatrix in Neurobasal medium/B27/L-glutamine for primary neuron culture, we achieved longer neurites (≥3,000 µm), greater cell viability (≥30%) for 2 months, and uniform culture across the wells. We also achieved an average neuronal ratio of 97%, showing a nearly pure culture of neurons without astrocytes. Our method is considerably better than techniques for the primary culture of neurons, and eliminates the need for a glial feeder layer. It also exhibits continued support for axonal elongation and synaptic activity for long periods (>6 weeks). 相似文献
8.
Insulin-like growth factor I has similar mitogenic effects to insulin, a growth factor required by most cells in culture,
and it can replace insulin in serum-free formulations for some cells. Chinese Hamster Ovary cells grow well in serum-free
medium with insulin and transferrin as the only exogenous growth factors. An alternative approach to addition of exogenous
growth factors to serum-free medium is transfection of host cells with growth factor-encoding genes, permitting autocrine
growth. Taking this approach, we constructed an IGF-I heterologous gene driven by the cytomegalovirus promoter, introduced
it into Chinese Hamster Ovary cells and examined the growth characteristics of Insulin-like growth factor I-expressing clonal
cells in the absence of the exogenous factor. The transfected cells secreted up to 500 ng/106 cells/day of mature Insulin-like growth factor I into the conditioned medium and as a result they grew autonomously in serum-free
medium containing transferrin as the only added growth factor. This growth-stimulating effect, observed under both small and
large scale culture conditions, was maximal since no further improvement was observed in the presence of exogenous insulin.
This revised version was published online in July 2006 with corrections to the Cover Date. 相似文献
9.
The derivation of human macrophages from peripheral blood monocytes remains a convenient method for the study of macrophage biology. However, for macrophage differentiation, a significant proportion of development has occurred prior to the monocyte stage; monocyte subsets also have varying potential for differentiation. Differentiation of macrophages from a less mature precursor, such as CD34+ haematopoietic stem cells, can further inform with regard to the development of macrophage-lineage cells. CD34+ cells were cultured in serum-free medium containing Flt3L, SCF, IL-3, IL-6 and M-CSF. Using differing combinations of growth factors, the effect on cell proliferation and differentiation to adherent macrophage-like cells was determined. The proliferative response of CD34+ cells to M-CSF was determined during the initial phase of cell culture. Thirteen combinations of SCF, IL-3, IL-6 and M-CSF were then compared to determine the optimum combination for proliferation. Adherence was used to isolate mature macrophages, and the macrophage-like phenotype was confirmed by analyses of surface markers, histo-morphology and phagocytosis. This study refines the means by which large numbers of macrophages are obtained under serum-free conditions from haematopoietic precursors. 相似文献
10.
Robert C. Callaghan Rosario Gil-Benso Antonio Pellin Antonio Llombart-Bosch 《Virchows Archiv. B, Cell pathology including molecular pathology》1991,60(1):271-278
This study examines the behavior of glycogenstoring rat hepatoma (N13) in vitro using cytophotometric techniques. A significant
increase in glycogen is observed in these cells after 30 min incubation in a buffered solution containing 0.1 mM glucose,
that is 80 times lower than the physiological glucose concentration in rat blood. N13 hepatoma cells grow exponentially in
culture using RPMI 1640 tissue culture medium supplemented with 10% fetal bovine serum. During the first day in culture these
cells store a large amount of glycogen and this increase is also observed in serum-free cultures. In more prolonged cultures
the amount of glycogen per cell gradually becomes lower, although the culturing conditions are maintained. Similar variations
of protein are also observed during the initial period of culture. DNA distribution does not show significant changes, although
in serum-free cultures an increase in the proportion of cells in S and G2/M phases is observed. The addition of glucagon, epinephrine and cyclic AMP derivatives to serum-free cultures does not impede
the storage of glycogen. Nevertheless, addition of either 2 mM N6,O2-dibutyryl cyclic AMP or 0.1 mM 8-(4-chlorophenylthio)-cyclic AMP blocks the cell cycle at G0/G1 and glycogen content does not decrease after the first day in culture. We believe that this cell line offers an appropriated
model to study glycogen metabolism and its involvement in the neoplastic process. 相似文献
11.
Kaori Ueda Akishi Onishi Shin-ichiro Ito Makoto Nakamura Masayo Takahashi 《Biochemical and biophysical research communications》2018,495(4):2595-2601
Purpose
Three-dimensional retinal organoids can be differentiated from embryonic stem cells/induced pluripotent stem cells (ES/iPS cells) under defined medium conditions. We modified the serum-free floating culture of embryoid body-like aggregates with quick reaggregation (SFEBq) culture procedure to obtain retinal organoids expressing more rod photoreceptors and S- and M-cone opsins.Methods
Retinal organoids differentiated from mouse Nrl-eGFP iPS cells were cultured in various mediums during photoreceptor development. To promote rod photoreceptor development, organoids were maintained in media containing 9-cis retinoic acids (9cRA). To obtain retinal organoids with M-opsin expression, we cultured in medium with 1% fetal bovine serum (FBS) supplemented with T3, BMP4, and DAPT. Section immunohistochemistry was performed to visualize the expression of photoreceptor markers.Results
In three-dimensional (3D) retinas exposed to 9cRA, rhodopsin was expressed earlier and S-cone opsins were suppressed. We could maintain 3D retinas up to DD 35 in culture media with 1% FBS. The 3D retinas expressed rhodopsin, S- and M-opsins, but most cone photoreceptors expressed either S- or M-opsins.Conclusion
By modifying culture conditions in the SFEBq protocol, we obtained rod-dominated 3D retinas and S- and M-opsin expressing 3D retinas. 相似文献12.
Osakada F Ikeda H Mandai M Wataya T Watanabe K Yoshimura N Akaike A Akaike A Sasai Y Takahashi M 《Nature biotechnology》2008,26(2):215-224
We previously reported the differentiation of mouse embryonic stem (ES) cells into retinal progenitors. However, these progenitors rarely differentiate into photoreceptors unless they are cultured with embryonic retinal tissues. Here we show the in vitro generation of putative rod and cone photoreceptors from mouse, monkey and human ES cells by stepwise treatments under defined culture conditions, in the absence of retinal tissues. With mouse ES cells, Crx+ photoreceptor precursors were induced from Rx+ retinal progenitors by treatment with a Notch signal inhibitor. Further application of fibroblast growth factors, Shh, taurine and retinoic acid yielded a greater number of rhodopsin+ rod photoreceptors, in addition to default cone production. With monkey and human ES cells, feeder- and serum-free suspension culture combined with Wnt and Nodal inhibitors induced differentiation of Rx+ or Mitf+ retinal progenitors, which produced retinal pigment epithelial cells. Subsequent treatment with retinoic acid and taurine induced photoreceptor differentiation. These findings may facilitate the development of human ES cell-based transplantation therapies for retinal diseases. 相似文献
13.
Routine culturing of normal,dysplastic and malignant human mammary epithelial cells from small tissue samples 总被引:5,自引:0,他引:5
Joanne T. Emerman Darcy A. Wilkinson 《In vitro cellular & developmental biology. Plant》1990,26(12):1186-1194
Summary We compared the growth and morphology of normal, dysplastic and malignant human mammary epithelial cells (HMEC) in medium
containing 5% human serum, a serum-free medium (32) and serum-free medium with a low Ca++ concentration. Tissues were dissociated and epithelial organoids or single cells were seeded onto collagen-coated dishes.
The cells grew in serum-containing medium, but growth of fibroblasts was also stimulated. The serum-free medium consistently
selected for and stimulated the growth of epithelial cells. There was little advantage in reducing the Ca++ concentration to further increase cell yield. This serum-free primary culture system allows us to routinely prouce sufficient
numbers of HMEC from small tissue samples for molecular biological investigations. Furthermore, the maintenance of cells in
a defined medium can provide a system for evaluating the direct effects of factors on gene expression.
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. 相似文献
14.
《Journal of Fermentation and Bioengineering》1994,77(4):436-438
A nicotinic acid-resistant mutant, designated NA201, was obtained from Fusarium sp. no. 5-128B by treatment with ultraviolet light. This mutant strain could grow in the presence of up to 500 mM nicotinic acid in the culture medium, although the parent strain could not grow at concentrations of nicotinic acid above 200 mM. The NA201 strain exhibited morphological mutations, neither forming aerial hyphae nor secreting a red-brown pigment. However, it retained the resistance to kabicidin at 25 mg l−t of the parent strain. The mutant NA201 cells contained high levels of squalene and low levels of ergosterol, about 53 times higher and five to six times lower, respectively, than those of the parent strain under standard culture conditions. The volumetric oxygen transfer coefficient (Kd) affected the level of squalene in the mutant cells. The Kd for the maximum production of squalene by the mutant was 24 mmol O2I−1h−1atm−1 and the level of squalene in the mutant cells was 26 mg (g cell)−1 on a dry weight basis. The greatest accumulation of squalene by the NA201 strain, corresponding to 323 mg per liter of culture medium and 35 mg (g cell)−1 on a dry weight basis, was achieved in a culture in which the Kd was changed from a high to a low value on the third day, with the simultaneous addition of 3% glucose (w/v). 相似文献
15.
《Cytotherapy》2022,24(8):850-860
Background aimsLentiviral vectors (LVs) have been used extensively in gene therapy protocols because of their high biosafety profile and capacity to stably express a gene of interest. Production of these vectors for the generation of chimeric antigen receptor (CAR) T cells in academic and research centers is achieved using serum-supplemented static monolayer cultures. Although efficient for pre-clinical studies, this method has a number of limitations. The main hurdles are related to its incompatibility with robust and controlled large-scale production. For this reason, cell suspension culture in bioreactors is desirable. Here the authors report the transition of LV particle production from serum-supplemented monolayer to serum-free suspension culture with the objective of generating CAR T cells.MethodsA self-inactivating LV anti-CD19 CAR was produced by transient transfection using polyethylenimine (PEI) in human embryonic kidney 293 T cells previously adapted to serum-free suspension culture.ResultsLV production of 8 × 106 transducing units (TUs)/mL was obtained in serum-supplemented monolayer culture. LV production in the serum-free suspension conditions was significantly decreased compared with monolayer production. Therefore, optimization of the transfection protocol was performed using design of experiments. The results indicated that the best condition involved the use of 1 μg of DNA/106 cells, 1 × 106 cells/mL and PEI:DNA ratio of 2.5:1. This condition used less DNA and PEI compared with the standard, thereby reducing production costs. This protocol was further improved with the addition of 5 mM of sodium butyrate and resulted in an increase in production, with an average of 1.5 × 105 TUs/mL. LV particle functionality was also assessed, and the results indicated that in both conditions the LV was capable of inducing CAR expression and anti-tumor response in T cells, which in turn were able to identify and kill CD19+ cells in vitro.ConclusionsThis study demonstrates that the transition of LV production from small-scale monolayer culture to scalable and controllable bioreactors can be quite challenging and requires extensive work to obtain satisfactory production. 相似文献
16.
《Journal of receptor and signal transduction research》2013,33(1-2):115-134
AbstractWe have identified one class of IGF-I-binding sites and two classes of IGF-II-binding sites at the surface of the melanoma cell line IGR39. By means of affinity labeling with 125I-IGF-I, a 290–300 kDa form was characterized. Using 125I-IGF-II, a 270 kDa polypeptide was labeled, corresponding to the type II IGF receptor. In the two serials of experiments, the order of potency in inhibiting 125I-IGF-I or 125I-IGF-II labeling of IGF-related peptides and αIR3, an antibody directed against type I receptor α subunit, was the same as in competition experiments. When IGR39 cells were cultured in a serum-free medium, the number of both high affinity IGF-II and IGF-I binding sites was increased, by 8-and 5-fold respectively, without any significant change in Kd values. In both culture conditions, we found IGFBP-2, -3, -4 and a 30 kDa form which Mr was consistent with IGFBP-5 or -6. Except for IGFBP-2, the amount of secreted IGFBPs was modified depending on culture conditions: in conditioned medium from cells cultured with 10% FCS, the amount of IGFBP-3 or -4 was higher, and the amount of the 30 kDa IGFBP was lower when compared to conditioned medium from cells cultured in serum-free medium. 相似文献
17.
Mouse-human hybridoma X87X cells were cultivated using a novel perfusion culture apparatus provided with three-settling zones to separate the cells from the culture medium by gravitational settling. The maximum viable cell density in a serum-free culture medium attained 3.0×107 cells/ml, when the specific perfusion rate was set to 2.3 vol day-1, and monoclonal antibody was continuously produced. These results were almost the same as those in the perfusion culture vessel with one settling zone and revealed that the process with a plurality of settling zones is a promising one for scale-up of a gravitation type of perfusion culture vessel. 相似文献
18.
《Journal of Fermentation and Bioengineering》1994,77(4):382-385
Production of 6-hydroxynicotinic acid, an important starting material for the synthesis of modern pesticides through bacterial position-specific hydroxylation of nicotinic acid, was investigated. Resting cells of Serratia marcescens IFO 12648 were found to catalyze the potential hydroxylation activity of nicotinic acid to produce 6-hydroxynicotinic acid. The optimum culture conditions of S. marcescens IFO 12648 for the accumulation of 6-hydroxynicotinic acid were investigated. The addition to the culture medium of molybdenum and iron ions and of nicotinic acid as an inducer greatly enhanced the hydroxylation activity. Under the optimum conditions, 98.5% of the added 2.2 M nicotinic acid was converted to 6-hydroxynicotinic acid, and the highest yield achieved was 301 g of 6-hydroxynicotinic acid per liter of reaction mixture containing 3.98 g dry weight of resting cells during a 72-h reaction at 35°C. 相似文献
19.
Hideki Agata Nobukazu Watanabe Noriyuki Kubo Mika Yamazaki Hideaki Kagami 《Biochemical and biophysical research communications》2009,382(2):353-155
Current standard techniques for bone tissue engineering utilize ex vivo expanded osteogenic cells. However, ex vivo expansion requires serum, which may hinder clinical applications. Here, we report the feasibility and efficacy of bone tissue engineering with human bone marrow stromal cells (BMSCs) expanded in serum-free conditions. Bone marrow was aspirated from 4 healthy donors and adherent cells were cultured in either serum-free medium (STEMPRO® MSC SFM) or conventional serum-containing medium (α-MEM supplemented with 10% serum). Efficacy of expansion was greater in serum-free medium. Phenotypically, serum-free expanded BMSCs were smaller in cell-size and showed expression of CD105++ and CD146dim. After osteogenic induction, serum-free expanded BMSCs showed lower alkaline phosphatase activity. However, they showed higher responsiveness to induction. In vivo bone-forming ability was also confirmed. In conclusion, bone tissue engineering with serum-free expanded BMSCs is feasible and as efficient as that obtained with BMSCs expanded in conventional serum-containing medium. 相似文献
20.
Xie C Jia B Xiang Y Wang L Wang G Huang G McNiece IK Wang J 《Cell and tissue research》2006,326(1):101-110
A novel indirect co-culture system was established to support ex vivo expansion of hematopoietic progenitors in umbilical cord blood (UCB) by using thrombopoietin (TPO)/Flt-3 ligand (FL)-transduced human-marrow-derived mesenchymal stem cells (tfhMSCs) as a feeder. UCB CD34+ cells were isolated and cultured by using five culture systems in serum-containing or serum-free medium. Suitable aliquots of cultured cells were taken to monitor cell production, clonogenic activity, and long-term culture-initiating culture (LTC-IC) output. Finally, the severe-combined immunodeficient mouse (SCID) repopulating cell (SRC) assay was performed to confirm the ability of the indirect co-cultured cells from the tfhMSCs system to reconstitute long-term hematopoiesis. Results showed significant differences in the number of total nucleated cells (TNCs) among the culture systems with respect to serum-containing medium or serum-free medium during 14-day culture. In addition, on day 14, the outputs of CD34+ cells, the colony-forming units (CFUs) in culture, and the CFUs in mixed colonies containing erythroid and myeloid cells and megakaryocytes in the tfhMSC indirect co-culture system were significantly enhanced. The LTC-IC assay demonstrated that the tfhMSCs indirect co-culture system had the strongest activity. The SCID-SRC assay confirmed the extensive ability of the expanded cells from the tfhMSCs indirect co-culture systems to reconstitute long-term hematopoiesis. Furthermore, polymerase chain reaction analysis demonstrated the presence of human hematopoietic cells in the bone marrow and peripheral blood cells of non-obese diabetic/SCID mice. Thus, hMSCs transduced with TPO/FL, in combination with additive cytokines, can effectively expand hematopoietic progenitors from UCB in vitro. The tfhMSC indirect co-culture system may therefore be a suitable system for ex vivo manipulation of primitive progenitor cells under non-contact culture conditions.This work was supported by the Zhejiang Scientific Foundation (no. 2003C23015). 相似文献