FBS-supplemented Media Research Articles

The Upscale Manufacture of Chondrocytes for Allogeneic Cartilage Therapies.

Allogeneic chondrocyte therapies need to be developed to allow more individuals to be treated with a cell therapy for cartilage repair and to reduce the burden and cost of the current two-stage autologous procedures. Upscale manufacture of chondrocytes using a bioreactor could help provide an off-the-shelf allogeneic chondrocyte therapy with many doses being produced in a single manufacturing run. In this study, we assess a good manufacturing practice-compliant hollow-fiber bioreactor (Quantum®) for adult chondrocyte manufacture. Chondrocytes were isolated from knee arthroplasty-derived cartilage (n = 5) and expanded in media supplemented with 10% fetal bovine serum (FBS) or 5% human platelet lysate (hPL) on tissue culture plastic (TCP) for a single passage. hPL-supplemented cultures were then expanded in the Quantum bioreactor for a further passage. Matched, parallel cultures in hPL or FBS were maintained on TCP. Chondrocytes from all culture conditions were characterized in terms of growth kinetics, morphology, immunoprofile, chondrogenic potential (chondrocyte pellet assays), and single telomere length analysis. Quantum expansion of chondrocytes resulted in 86.4 ± 38.5 × 106 cells in 8.4 ± 1.5 days, following seeding of 10.2 ± 3.6 × 106 cells. This related to 3.0 ± 1.0 population doublings in the Quantum bioreactor, compared with 2.1 ± 0.6 and 1.3 ± 1.0 on TCP in hPL- and FBS-supplemented media, respectively. Quantum- and TCP-expanded cultures retained equivalent chondropotency and mesenchymal stromal cell marker immunoprofiles, with only the integrin marker, CD49a, decreasing following Quantum expansion. Quantum-expanded chondrocytes demonstrated equivalent chondrogenic potential (as assessed by ability to form and maintain chondrogenic pellets) with matched hPL TCP populations. hPL manufacture, however, led to reduced chondrogenic potential and increased cell surface positivity of integrins CD49b, CD49c, and CD51/61 compared with FBS cultures. Quantum expansion of chondrocytes did not result in shortened 17p telomere length when compared with matched TCP cultures. This study demonstrates that large numbers of adult chondrocytes can be manufactured in the Quantum hollow-fiber bioreactor. This rapid, upscale expansion does not alter chondrocyte phenotype when compared with matched TCP expansion. Therefore, the Quantum provides an attractive method of manufacturing chondrocytes for clinical use. Media supplementation with hPL for chondrocyte expansion may, however, be unfavorable in terms of retaining chondrogenic capacity.

Enhancing Mucosal-Associated Invariant T Cell Function and Expansion with Human Selective Serum.

Mucosal-associated invariant T (MAIT) cells are promising innate-like lymphocytes with potential for use in anti-tumor immunotherapy. Existing MAIT cell expansion protocols are associated with potentially decremental phenotypic changes, including increased frequency of CD4+ MAIT cells and higher inhibitory receptor expression. In this study, we compared the effect on expansion of human MAIT cells of a serum replacement, Physiologix XF SR (Phx), with traditional serum FBS for supplementing RPMI 1640 media. Using flow cytometry, we found that Phx supported a significantly higher proliferative capacity for MAIT cells and resulted in a lower frequency of CD4+ MAIT cells, which have been associated with reduced Th1 effector and cytolytic functions. We saw that culturing MAIT cells in Phx led to better survival of MAIT cells and lower frequency of PD-1+ MAIT cells than FBS-supplemented media. Functionally, we saw that Phx supplementation was associated with a higher frequency of IFN-γ+ MAIT cells after stimulation with Escherichia coli than FBS-supplemented RPMI. In conclusion, we show that MAIT cells cultured in Phx have higher proliferative capacity, lower expression of inhibitory receptors, and higher capacity to produce IFN-γ after E. coli stimulation than FBS-supplemented RPMI. This work shows that expanding MAIT cells with Phx compared with FBS-supplemented RPMI results in a more functionally desirable MAIT cell for future anti-tumor immunotherapy.

Endogenous cell-free DNA in fetal bovine serum introduces artifacts to in vitro cell-free DNA models.

Cell-free DNA (cfDNA) is of growing clinical and research significance. In vitro cfDNA models are a useful tool in cfDNA research; however, artifacts in these models may have implications for the interpretation of new and published data. This report aimed to establish how endogenous cfDNA in fetal bovine serum (FBS) may influence in vitro cfDNA measurements. Three commercial cell culture media, supplemented with 10% FBS, were analyzed for the presence of cfDNA, with and without culture with ovarian cancer cell lines. cfDNA from FBS was identified with all three commercial media and contributed a major portion of 167-bp cfDNA. Future studies should account for bovine cfDNA in FBS-supplemented media when conducting in vitro cfDNA research.

Investigating the Impact of Serum on the Growth of TM7x and Host XH001

Nanosynbacter lyticus strain TM7x is an ultra-small, parasitic bacterium that lives on the surface of host, Actinomyces odontolyticus strain XH001. Studies have reported a considerable increase in TM7x abundance in patients with periodontal disease, and this association warrants further investigation. Interestingly, gingival crevicular fluid (GCF) flow also increases during periodontitis. The prevalence of TM7x during periodontal disease may thus result from attraction to GCF. However, GCF is not commercially available for testing. Since its composition is similar to that of fetal bovine serum (FBS), FBS was tested instead. The objectives of this project are to determine whether FBS promotes the growth of TM7x and XH001 and to develop the optimal culture medium for analysis of these microbes. TM7x/XH001 co-culture and XH001 monoculture were separately cultivated in media (RPMI and FMC) supplemented with FBS. Cultures grown in the same, non-supplemented media served as control samples. Serial dilutions were performed and spotted onto plates at several time points, and samples were quantified in colony-forming units (CFU)/mL. 2% FBS FMC induced a significant increase in the growth of culture samples, as compared to control FMC (p = 0.0140 and p = 0.0424 for the co-culture and monoculture, respectively). Furthermore, microscopy revealed a heightened presence of TM7x in FBS-supplemented media in comparison to control media. These findings signify that FBS is an effective supplement to RPMI and FMC, both of which support TM7x/XH001 growth. Overall, the utilization of these media with FBS can ensure optimal cultivation of TM7x for investigation of its potential pathogenicity.

Cellular uptake and toxicity of gold nanoparticles on two distinct hepatic cell models

Gold nanoparticles (AuNPs) have huge potential for various biomedical applications, but their successful use depends on their uptake and possible toxicity in the liver, their main site for accumulation. Therefore, in this work we compared the cytotoxic effects induced by AuNPs with different size (~ 15 nm and 60 nm), shape (nanospheres and nanostars) and capping [citrate- or 11-mercaptoundecanoic acid (MUA)], in human HepaRG cells or primary rat hepatocytes (PRH) cultivated with serum-free or Foetal Bovine Serum (FBS)-supplemented media.The safety assessment of the AuNPs demonstrated that overall they present low toxicity towards hepatic cells. Among all the tested AuNPs, the smaller 15 nm spheres displayed the highest toxicity. The toxicological effect was capping, size and cell-type dependent with citrate-capping more toxic than MUA (PRH with FBS), the 15 nm AuNPs more toxic than 60 nm counterparts and PRH more sensitive, as compared to the HepaRG cells. The incubation with FBS-free media produced aggregation of AuNPs while its presence greatly influenced the toxicity outcomes. The cellular uptake of AuNPs was shape, size and capping dependent in PRH cultivated in FBS-supplemented media, and significantly different between the two types of cells with extensively higher internalization of AuNPs in PRH, as compared to the HepaRG cells.These data show that the physical-chemical properties of AuNPs, including size and shape, as well as the type of cellular model, greatly influence the interaction of the AuNPs with the biological environment and consequently, their toxicological effects.

Human Embryonic-Derived Mesenchymal Progenitor Cells (hES-MP Cells) are Fully Supported in Culture with Human Platelet Lysates.

Human embryonic stem cell-derived mesenchymal progenitor (hES-MP) cells are mesenchymal-like cells, derived from human embryonic stem cells without the aid of feeder cells. They have been suggested as a potential alternative to mesenchymal stromal cells (MSCs) in regenerative medicine due to their mesenchymal-like proliferation and differentiation characteristics. Cells and cell products intended for regenerative medicine in humans should be derived, expanded and differentiated using conditions free of animal-derived products to minimize risk of animal-transmitted disease and immune reactions to foreign proteins. Human platelets are rich in growth factors needed for cell culture and have been used successfully as an animal serum replacement for MSC expansion and differentiation. In this study, we compared the proliferation of hES-MP cells and MSCs; the hES-MP cell growth was sustained for longer than that of MSCs. Growth factors, gene expression, and surface marker expression in hES-MP cells cultured with either human platelet lysate (hPL) or fetal bovine serum (FBS) supplementation were compared, along with differentiation to osteogenic and chondrogenic lineages. Despite some differences between hES-MP cells grown in hPL- and FBS-supplemented media, hPL was found to be a suitable replacement for FBS. In this paper, we demonstrate for the first time that hES-MP cells can be grown using platelet lysates from expired platelet concentrates (hPL).

Nanofiltration of growth media supplemented with human platelet lysates for pathogen-safe xeno-free expansion of mesenchymal stromal cells

Background aimsHuman platelet lysate can replace fetal bovine serum (FBS) for xeno-free ex vivo expansion of mesenchymal stromal cells (MSCs), but pooling of platelet concentrates (PCs) increases risks of pathogen transmission. We evaluated the feasibility of performing nanofiltration of platelet lysates and determined the impact on expansion of bone marrow–derived MSCs. MethodsPlatelet lysates were prepared by freeze-thawing of pathogen-reduced (Intercept) PCs suspended in 65% storage solution (SPP+) and 35% plasma, and by serum-conversion of PCs suspended in 100% plasma. Lysates were added to the MSC growth media at 10% (v/v), filtered and subjected to cascade nanofiltration on 35- and 19-nm Planova filters. Media supplemented with 10% starting platelet lysates or FBS were used as the controls. Impacts of nanofiltration on the growth media composition, removal of platelet extracellular vesicles (PEVs) and MSC expansion were evaluated. ResultsNanofiltration did not detrimentally affect contents of total protein and growth factors or the biochemical composition. The clearance factor of PEVs was >3 log values. Expansion, proliferation, membrane markers, differentiation potential and immunosuppressive properties of cells in nanofiltered media were consistently better than those expanded in FBS-supplemented media. Compared with FBS, chondrogenesis and osteogenesis genes were expressed more in nanofiltered media, and there were fewer senescent cells over six passages. ConclusionsNanofiltration of growth media supplemented with two types of platelet lysates, including one prepared from pathogen-reduced PCs, is technically feasible. These data support the possibility of developing pathogen-reduced xeno-free growth media for clinical-grade propagation of human cells.

Towards a consensus potency assay for mesenchymal stromal cells: identification of activation markers reliable across media formulations, donor and tissue source

Background & Aim Reliable metrics to assess therapeutic potential of mesenchymal stromal cell (MSC) batches are needed for successful translation of MSC-based products. We previously reported that 1 of 47 ISCT-proposed surrogate markers of immunomodulation, ICAM-1, reports activation in 3 donor populations each of human umbilical cord (UC) and bone marrow (BM) MSCs primed by TNF-α, IL-1β or IFN-γ. Seventeen other transcript markers are reliable in at least 1 context. Here we expanded the test matrix to include protein-free (PF), xeno-free (XF) and FBS supplemented (FS) media, and combined TFN-α+IFN-γ. Methods, Results & Conclusion UC-MSCs (Tissue Regeneration Therapeutics Inc., CAN) and BM-MSCs (RoosterBio Inc. (RBI), USA) were cultured in XF or FS media (RBI). MSCs were primed for 24 hours with TNF-α (50ng/ml), IFN-γ (50ng/ml), IL-1β (80pg/ml) or TNF-α+IFN-γ (10ng/ml each) in PF, XF or FS media (n=3). RT-qPCR was performed for the 18 curated ISCT genes and 3 potential new markers identified in the first study. Data was normalized to mean expression of ACTB, GAPDH, RPS18 and YWHAZ reference genes (ΔCq), and relative RNA abundance of primed versus resting MSCs assessed using 2−ΔΔCq. Statistically significant differences were determined by false discovery rate-adjusted p The refined panel of surrogate markers was responsive in all 3 media formulations within the test matrices, although relative levels of 9 biomarkers were significantly affected by media type in at least 1 context. Notably, ICAM-1 and 1 candidate gene remained reliable in all conditions. Stimulation with TFN-α+IFN-γ can synergistically enhance MSC immune-modulatory potency. Here, only CCL5, CXCL11, IL-6 and TNFSF10 reflected this phenomenon with a synergistic increase in transcript levels in all media types, although IL-6 increased more in BM-MSCs. Intriguingly, CXCL8 and HLA-DRB1 specifically decreased in FS media, while CIITA and HLA-DRA decreased in both supplemented formulations but not in PF media. CCL2 was the only reduced transcript in PF media, although it synergistically increased in FS media. The 3 putative activation biomarkers also synergistically increased in XF and FS media treated with TFN-α+IFN-γ. Taken together, we have identified ICAM-1 and a new gene marker as suitable candidates for further development of standardized MSC performance assays. Ongoing work seeks to correlate levels of surrogate markers to clinical outcomes and define thresholds that functionally qualify MSC batches.

The influence of serum-supplemented culture media in a transwell migration assay.

Our work cautions against the use of serum-supplemented culture media in a transwell migration assay when using chemoattractants other than FBS. At 24 h, a 5% foetal bovine serum (FBS) gradient caused BV2 microglia to migrate toward the lower compartment of the transwell apparatus. Interestingly, FBS-supplemented media in the absence of a gradient also resulted in notable microglia migration. Serum can therefore confound the interpretation of a transwell migration assay when another chemoattractant is used.

Human serum enhances the proliferative capacity and immunomodulatory property of MSCs derived from human placenta and umbilical cord

BackgroundMesenchymal stromal cells (MSCs) are considered potential candidates that hold great promise in the treatment of immune-related diseases. For therapeutic applications, it is necessary to isolate and expand MSCs with procedures complying with good manufacturing practice (GMP). Recent studies reported the use of human serum (HS) instead of fetal bovine serum (FBS) for the expansion of bone marrow-derived MSCs. Nevertheless, there are only limited data on HS as an alternative to FBS for the isolation and expansion of umbilical (UC-MSCs) and placenta-derived MSCs (PL-MSCs). In this study, we evaluate the effect of HS compared to FBS on the proliferative and immunosuppressive capacities of these MSCs.MethodsPL-MSCs and UC-MSCs were isolated and cultured in HS- or FBS-supplemented media. The MSC characteristics, including morphology, immunophenotype, and differentiation ability, were verified. The proliferative and immunosuppressive capacities were also examined. In addition, the proliferative-enhancing factors in both sera were explored using proteomic analysis.ResultsPL-MSCs and UC-MSCs proliferated faster in HS-supplemented medium than in equivalent levels of FBS-supplemented medium. Adipogenic and osteogenic differentiations occurred at nearly identical levels in HS- and FBS-supplemented media. Interestingly, MSCs cultured in HS-supplemented medium had a similar immunosuppressive effect as MSCs cultured in FBS-supplemented medium. Proteomic analysis revealed that Con-A binding glycoproteins with a molecular weight > 100 kDa in FBS could significantly enhance MSC proliferation. In contrast, the proliferative enhancing factors in HS were found in the Con-A non-binding fraction and WGA binding fraction with a molecular weight > 100 kDa.ConclusionsTaken together, our results suggest applications for the use of HS instead of FBS for the isolation and expansion of PL-MSCs and UC-MSCs for cell therapy in the future. Furthermore, this study identifies factors in HS that are responsible for its proliferative and immunosuppressive effects and might thus lead to the establishment of GMPs for the therapeutic use of MSCs.

Angiogenic effect of platelet-rich concentrates on dental pulp stem cells in inflamed microenvironment.

In this study, we aimed to determine the suitable concentrations of human platelet lysate (HPL) and platelet-rich plasma (PRP) for maintaining the in vitro proliferative and angiogenic potential of inflamed dental pulp stem cells. Lipopolysaccharide (LPS)-induced inflamed dental pulp-derived stem cells (iDPSCs) were treated with different concentrations of HPL and PRP (10% and 20%) followed by determination of viability using Alamar Blue assay. Expression of angiogenesis-, adhesion-, and inflammation-regulating genes was also analyzed using RT-qPCR array. Furthermore, expression of growth factors at protein level in the cell culture microenvironment was measured using multiplex assay. Viability of iDPSCs was significantly (p < 0.05) higher in 20% HPL-supplemented media compared to iDPSCs. Expression of 10 out of 12 selected angiogenic genes, four out of seven adhesion molecules, and seven out of nine cytokine-producing genes were significantly (p < 0.05) higher in cells maintained in 20% HPL-supplemented media compared to that in FBS-supplemented media. Furthermore, expression of all the selected growth factors was significantly higher (p < 0.05) in the supernatants from 20% HPL media at 12 and 24h post-incubation. This study suggests that 20% HPL could be optimum to stimulate angiogenesis-related factors in iDPSCs while maintaining their viability. This data may suggest the potential use of 20% HPL for expanding DPSCs scheduled for clinical trials for regenerative therapies including dental pulp regeneration.

Efficacy of Humanized Mesenchymal Stem Cell Cultures for Bone Tissue Engineering: A Systematic Review with a Focus on Platelet Derivatives.

Fetal bovine serum (FBS) is the most commonly used supplement for ex vivo expansion of human mesenchymal stem cells (hMSCs) for bone tissue engineering applications. However, from a clinical standpoint, it is important to substitute animal-derived products according to current good manufacturing practice (cGMP) guidelines. Humanized alternatives to FBS include three categories of products: human serum (HS), human platelet derivatives (HPDs)-including platelet lysate (PL) or platelet releasate (PR), produced by freeze/thawing or chemical activation of platelet concentrates, respectively, and chemically defined media (serum-free) (CDM). In this systematic literature review, the in vitro and in vivo osteogenic potential of hMSCs expanded in humanized (HS-, HPD-, or CDM-supplemented) media versus hMSCs expanded in FBS-supplemented media, was compared. In addition, PL and PR were compared in terms of their growth factor (GF)/cytokine-content and cell-culture efficacy. When using either 10-20% autologous or pooled HS, 3-10% pooled HPDs or CDM supplemented with GFs, in comparison with 10-20% FBS, a majority of studies reported similar or superior in vitro proliferation and osteogenic differentiation, and in vivo bone formation in ectopic or orthotopic rodent models. Moreover, a trend for higher GF content was observed in PL versus PR, although evidence for cell culture efficacy is limited. In summary, humanized supplements seem at least equally effective as FBS for hMSC expansion and osteogenic differentiation. Although pooled HPDs appear to be the most favorable supplement for large-scale hMSC expansion, further efforts are needed to standardize the preparation and composition of these products in compliance with cGMP standards.

Biocompatible polymeric coatings do not inherently reducethe cytotoxicity of iron oxide nanoparticles

Nanotechnology in biomedical research is an emerging and promising tool for different purposes, like high-resolution medical imaging, diagnostics, and targeted drug delivery. Although some experimental efforts focused on determination of the cytotoxicity of nanoparticles (NPs) are present, there are many controversial results. In this study, chitosan (CS)- and poly(acrylic acid) (PAA)-coated iron oxide nanoparticles (IONPs) were used to investigate the possible cytotoxicity of coated IONPs on model mammalian cell line SaOs-2 by evaluating cellular viability and membrane integrity. Increasing concentrations of IONPs increased the cytotoxic effect on SaOs-2 cells with both CS- and PAA-coated IONPs. Cell viability on day 3 was as low as 40% and 48% at 1000 ?M concentration for PAAand CS-coated IONPs, respectively, in 1% FBS-supplemented media. Cytotoxicity determined by the released lactate dehydrogenase (LDH) was as high as 163% with 1000 ?M concentration of CS-IONPs, while there was no significant change in LDH release with PAAcoated IONPs at any concentration. This study reveals that IONPs coated with a biocompatible polymer, which are usually assumed to be nontoxic, show cytotoxicity with increasing concentration and incubation time. The cytotoxicity of nanoparticles intended for biomedical purposes must be evaluated using more than one approach.

Canine Platelet Lysate Is Inferior to Fetal Bovine Serum for the Isolation and Propagation of Canine Adipose Tissue- and Bone Marrow-Derived Mesenchymal Stromal Cells.

BackgroundMesenchymal stromal cells (MSC) are increasingly investigated for their clinical utility in dogs. Fetal bovine serum (FBS) is a common culture supplement used for canine MSC expansion. However, FBS content is variable, its clinical use carries risk of an immune response, and its cost is increasing due to global demand. Platelet lysate (PL) has proven to be a suitable alternative to FBS for expansion of human MSC.Hypothesis and ObjectivesWe hypothesized that canine adipose tissue (AT) and bone marrow (BM) MSC could be isolated and expanded equally in PL and FBS at conventionally-used concentrations with differentiation of these MSC unaffected by choice of supplement. Our objectives were to evaluate the use of canine PL in comparison with FBS at four stages: 1) isolation, 2) proliferation, 3) spontaneous differentiation, and 4) directed differentiation.Results1) Medium with 10% PL was unable to isolate MSC. 2) MSC, initially isolated in FBS-supplemented media, followed a dose-dependent response with no significant difference between PL and FBS cultures at up to 20% (AT) or 30% (BM) enrichment. Beyond these respective peaks, proliferation fell in PL cultures only, while a continued dose-dependent proliferation response was noted in FBS cultures. 3) Further investigation indicated PL expansion culture was inducing spontaneous adipogenesis in concentrations as low as 10% and as early as 4 days in culture. 4) MSC isolated in FBS, but expanded in either FBS or PL, maintained ability to undergo directed adipogenesis and osteogenesis, but not chondrogenesis.Conclusions/SignificanceCanine PL did not support establishment of MSC colonies from AT and BM, nor expansion of MSC, which appear to undergo spontaneous adipogenesis in response to PL exposure. In vivo studies are warranted to determine if concurrent use of MSC with any platelet-derived products such as platelet-rich plasma are associated with synergistic, neutral or antagonistic effects.

Effect of serum choice on replicative senescence in mesenchymal stromal cells

Background aimsMultipotent mesenchymal stromal cells (MSCs) are promising candidates for innovative cell therapeutic applications. Before their use, however, they usually need to be expanded in vitro with serum-supplemented media. MSCs can undergo replicative senescence during in vitro expansion, but it is not yet clear how serum supplements influence this process. MethodsIn the present study, we compared how media supplemented with fetal bovine serum (FBS) or calf serum (CS) affected morphology, proliferation, differentiation, senescence and other functional characteristics of human umbilical cord–derived MSCs (UC-MSCs). ResultsUC-MSCs cultured in both FBS- and CS-containing media were able to differentiate along osteogenic and adipogenic lineages but ultimately reached proliferation arrest. However, senescence-associated characteristics, such as β-galactosidase activity, reactive oxygen species levels, proliferation rate and gene expression, demonstrate that UC-MSCs grown with FBS have better proliferation potential and differentiation capacity. In contrast, UC-MSCs grown with CS have a higher proportion of apoptotic cells and senescent characteristics. Possible mechanisms for the observed phenotypes include changes in gene expression (Bax, p16, p21 and p53) and cytokine production (interleukin-6 and interleukin-8). ConclusionsThis study demonstrates that FBS-supplemented media provides a better microenvironment for the expansion of UC-MSCs in vitro than CS-supplemented media. This work provides insight into MSCs generation practices for use in basic research and clinical therapies.

The effects of human serum to the morphology, proliferation and gene expression level of the respiratory epithelium in vitro

The culture of human airway epithelial cells has played an important role in advancing our understanding of the metabolic and molecular mechanisms underlying normal function and disease pathology of airway epithelial cells. The present study focused on investigating the effects of human serum (HS) on the qualitative and quantitative properties of the human respiratory epithelium compared to the fetal bovine serum (FBS), as a supplement in culture. Respiratory epithelial (RE) cells derived from human nasal turbinate were co-cultured with fibroblasts, subsequently separated at 80-90% confluency by differential trypsinization. RE cells were then sub-cultured into 2 different plates containing 5% allogenic HS and FBS supplemented media respectively up to passage 1 (P1). Cell morphology, growth rate, cell viability and population doubling time were assessed under light microscope, and levels of gene expression were measured via real time reverse transcriptase-polymerase chain reaction (qRT-PCR). RE cells appeared as polygonal shape and expanded when cultured in HS whereas RE cells in FBS were observed to be easily matured thus limit the RE cells expansion. Proliferation rate of RE cells in HS supplemented media (7673.18 ± 1207.15) was 3 times higher compared to RE in FBS supplemented media (2357.68 ± 186.85). Furthermore, RE cells cultured in HS-supplemented media required fewer days (9.15 ± 1.10) to double in numbers compared to cells cultured in FBS-supplemented media (13.66 ± 0.81). Both the differences were significant (p<0.05). However, there were no significant differences in the viability of RE cells in both groups (p=0.105). qRT-PCR showed comparable expressions of gene Cytokeratin-14 (CK-14), Cytokeratin-18 (CK-18) and Mucin-5 subtype B (MUC5B) in RE cells cultured in both groups (p>0.05). In conclusion, HS is a comparatively better choice of media supplement in accelerating growth kinetics of RE cells in vitro thus producing a better quality of respiratory epithelium for future tracheal reconstruction.

Transplantation of Autologous Serum-Derived Cultivated Corneal Epithelial Equivalents for the Treatment of Severe Ocular Surface Disease

To evaluate the use of autologous serum (AS)-derived cultivated corneal epithelial transplantation for the treatment of severe ocular surface disease. Retrospective noncomparative case series. Nine eyes from 9 patients with total limbal stem cell deficiency were studied. These consisted of 2 eyes with Stevens-Johnson syndrome, 1 with chemical injury, 1 with ocular cicatricial pemphigoid, 1 with Salzmann corneal dystrophy, 1 with aniridia, 1 with graft-versus-host disease, and 2 with idiopathic ocular surface disease. Autologous serum obtained from patients was used for cultivating corneal epithelial cells on an amniotic membrane substrate. These AS-derived corneal epithelial equivalents were compared with those derived from fetal bovine serum (FBS)-supplemented medium. At the time of surgery, complete removal of the corneal pannus and conjunctiva up to 3 mm from the limbus was performed. Allogeneic (7 cases) and autologous (2 cases) AS-derived cultivated corneal epithelial equivalents were transplanted onto the ocular surface. Postoperative follow-up included serial slit-lamp examinations with fluorescein staining, as well as photographic documentation. Ocular surface reconstruction with corneal epithelialization, graft integrity, visual acuity, and postoperative complications. The corneal epithelial sheets cultivated in AS- and FBS-supplemented media were morphologically similar, and demonstrated the normal expression of tissue-specific keratins and junctional specialization assembly proteins. After transplantation, complete corneal epithelialization was achieved within 2 to 5 days. All eyes demonstrated an improvement in visual acuity by > or =2 lines. During the follow-up period of 14.6+/-4.36 months, the corneal surface of all patients remained stable and transparent, without significant complications. Transplantation of AS-derived cultivated corneal epithelial equivalents was shown to be a feasible method of treating patients with severe ocular surface disease. The use of AS is of clinical importance in the development of autologous xenobiotic-free bioengineered ocular surface equivalents for clinical transplantation.

The Use of Autologous Serum in the Development of Corneal and Oral Epithelial Equivalents in Patients with Stevens-Johnson Syndrome

To evaluate the use of autologous serum (AS) from patients with severe ocular surface disease (OSD) in the development of transplantable corneal and oral epithelial tissue equivalents and to compare it with the use of conventional culture methods by using fetal bovine serum (FBS). AS was obtained from patients with severe OSD secondary to Stevens-Johnson syndrome. Corneal and oral epithelial cells were cultivated in medium supplemented with either AS or FBS. Corneal and oral epithelial equivalents were constructed on denuded amniotic membranes. The bromodeoxyuridine (BrdU) ELISA cell proliferation assay and colony-forming efficiency (CFE) of cells cultivated in AS- or FBS-supplemented media were compared. The morphologic characteristics and the basement membrane assembly of cultivated epithelial equivalents were analyzed by light and electron microscopy, as well as by immunohistochemistry. BrdU proliferation assay and CFE analysis showed that human corneal and oral epithelial cells cultivated in AS-supplemented media had comparable proliferative capacities compared with FBS-supplemented media. The corneal and oral epithelial equivalents cultivated in AS- and FBS-supplemented media were morphologically similar and demonstrated the normal expression of tissue-specific keratins and basement membrane assembly. The presence of a well-formed stratified epithelium, a basement membrane, and hemidesmosomal attachments was confirmed by electron microscopy. AS-supplemented cultures were effective in supporting the proliferation of human corneal and oral epithelial cells, as well as the development of transplantable epithelial equivalents. The use of AS is of clinical importance in the development of autologous xenobiotic-free bioengineered ocular surface equivalents for clinical transplantation.

Use of plant‐derived protein hydrolysates for enhancing growth ofBombyx mori(silkworm) insect cells in suspension culture

The successful suspension culture of the Bombyx mori (silkworm) cell lines Bm5 and BmN4 without FBS (fetal-bovine serum) was first realized in Sf-900 II SFM (serum-free medium) (Gibco BRL, Rockville, MD, U.S.A.) supplemented with a plant-derived protein hydrolysate. The addition of 0.5% HyPep 1510 (Difco Co., Detroit, MI, U.S.A.) and 10 mM glutamine to Sf-900 II SFM at 4 days of culture was found effective in increasing the cell concentration to 8.5x10(6) cells/ml. The replacement of medium with Sf-900 II SFM supplemented with 0.5% HyPep 1510 at 6 days of culture increased the cell concentration by 1.1x10(7) cells/ml. When Sf-900 II SFM was supplemented with 0.5% Hypep 1510, 16 days, which was half of the conventional adaptation time, was sufficient for the B. mori cell line to adapt to SFM and shear stress while maintaining a stable viability. The beta-galactosidase activity in Sf-900 II SFM supplemented with 0.5% Hypep 1510 was 4.9x10(3) units/ml, which was 2-fold higher than that of the FBS-supplemented medium. By SDS/PAGE, only the band corresponding to beta-galactosidase was detected in the sample from the media supplemented with plant-derived protein hydrolysates, while thick bands corresponding to proteins having lower molecular masses than beta-galactosidase were detected in samples from the FBS-supplemented media. These results suggest that plant-derived protein hydrolysates are promising FBS substitutes for enhancing the growth of B. mori cells and facilitating the purification of recombinant proteins produced by baculovirus infection.

Equiguard™ suppresses androgen-dependent LNCaP prostate cancer cell proliferation by targeting cell cycle control via down regulation of the retinoblastoma protein Rb and induction of apoptosis via the release of cytochrome c

Lack of life-prolonging therapies has provided much of the impetus for seeking complementary and alternative management/treatment options by prostate cancer (CaP) patients. Among these, the use of dietary supplements and botanical products has been showing a sustained increase in recent years, owing in part to some encouraging pre-clinical and clinical data shown in a limited number of herbal products. Notably, however, the majority of herbal and dietary supplement products have not been rigorously studied with regard to their efficacy. In vitro mechanistic experiments are considered essential preludes and requisites to more lengthy and costly animal and human studies, in that they may provide relevant insights and scientific basis for effects some of these products purportedly might demonstrate. In vitro studies in our laboratory have shown that a polyherbal supplement, Equiguard, exhibits anti-tumor activity against hormone dependent LNCaP cells cultured in both androgen-proficient (FBS) and -deficient (CS-FBS) conditions. Clinically relevant anti-prostate cancer effects of Equiguard are vividly illustrated by growth suppression and down regulated expression of prostate specific genes, respectively, androgen receptor (AR) and prostate specific antigen (PSA). However, the mechanistic bases contributing to these effects have not been well characterized. This communication describes experiments aimed at further understanding growth arrest elicited by Equiguard in LNCaP cells cultured in FBS and CS-FBS conditions. We have focused on aspects of cell cycle control and induction of apoptosis. Regulation of cell cycle progression by Equiguard was analyzed by examining changes in the expression of Rb and cyclins D/E. Using Western blot analysis, we showed that treatment caused inhibition of Rb phosphorylation, which was accompanied by the reduction of cyclins D/E expression, in both culture conditions. Moreover, cells treated with Equiguard and cultured with FBS-supplemented media showed up-regulation of cyclin-dependent kinase inhibitor Kip1/p27. These results support the interpretation that suppression of Rb phosphorylation mediated the observed growth arrest induced by Equiguard under androgen-proficient condition. In contrast, Equiguard-treated cells cultured in CS-FBS had lowered expression of the Kip1/p27, suggesting that different control mechanisms, possibly evoked by changes in cellular microenvironments, contributed to growth suppression by Equiguard. The growth suppressive effects of Equiguard in both culture conditions were also evaluated with respect to induction of apoptosis. While Equiguard elicited apoptosis was accompanied by an increase in the level of cytosolic cytochrome c, the relative accumulation of cytochrome c in the cytosol was unaffected by culture conditions. These results suggest that the ability to trigger apoptosis as one aspect of the control of cell growth by Equiguard is integrally linked to the release of cytochrome c, by a mechanism largely independent of the presence of androgens.