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1.
Stem Cell Res Ther ; 12(1): 506, 2021 09 16.
Artigo em Inglês | MEDLINE | ID: mdl-34530920

RESUMO

BACKGROUND: Mesenchymal stromal cells (MSCs) are a potential therapeutic tool for pulmonary fibrosis. However, ex vivo MSC expansion using serum poses risks of harmful immune responses or unknown pathogen infections in the recipients. Therefore, MSCs cultured in serum-free media (SF-MSCs) are ideal for clinical settings; however, their efficacy in pulmonary fibrosis is unknown. Here, we investigated the effects of SF-MSCs on bleomycin-induced pulmonary inflammation and fibrosis compared to those of MSCs cultured in serum-containing media (S-MSCs). METHODS: SF-MSCs and S-MSCs were characterized in vitro using RNA sequence analysis. The in vivo kinetics and efficacy of SF-MSC therapy were investigated using a murine model of bleomycin-induced pulmonary fibrosis. For normally distributed data, Student's t test and one-way repeated measures analysis of variance followed by post hoc Tukey's test were used for comparison between two groups and multiple groups, respectively. For non-normally distributed data, Kruskal-Wallis and Mann-Whitney U tests were used for comparison between groups, using e Bonferroni's correction for multiple comparisons. All tests were two-sided, and P < 0.05 was considered statistically significant. RESULTS: Serum-free media promoted human bone marrow-derived MSC expansion and improved lung engraftment of intravenously administered MSCs in recipient mice. SF-MSCs inhibited the reduction in serum transforming growth factor-ß1 and the increase of interleukin-6 in both the serum and the bronchoalveolar lavage fluid during bleomycin-induced pulmonary fibrosis. SF-MSC administration increased the numbers of regulatory T cells (Tregs) in the blood and lungs more strongly than in S-MSC administration. Furthermore, SF-MSCs demonstrated enhanced antifibrotic effects on bleomycin-induced pulmonary fibrosis, which were diminished by antibody-mediated Treg depletion. CONCLUSIONS: SF-MSCs significantly suppressed BLM-induced pulmonary inflammation and fibrosis through enhanced induction of Tregs into the lungs and corrected the dysregulated cytokine balance. Therefore, SF-MSCs could be a useful tool for preventing pulmonary fibrosis progression without the demerits of serum use.


Assuntos
Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais , Fibrose Pulmonar , Animais , Bleomicina/toxicidade , Medula Óssea , Células Cultivadas , Meios de Cultura Livres de Soro , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Fibrose Pulmonar/induzido quimicamente , Fibrose Pulmonar/terapia , Linfócitos T Reguladores
2.
Elife ; 102021 09 29.
Artigo em Inglês | MEDLINE | ID: mdl-34586063

RESUMO

Lung epithelial progenitors differentiate into alveolar type 1 (AT1) and type 2 (AT2) cells. These cells form the air-blood interface and secrete surfactant, respectively, and are essential for lung maturation and function. Current protocols to derive and culture alveolar cells do not faithfully recapitulate the architecture of the distal lung, which influences cell fate patterns in vivo. Here, we report serum-free conditions that allow for growth and differentiation of mouse distal lung epithelial progenitors. We find that Collagen I promotes the differentiation of flattened, polarized AT1 cells. Using these organoids, we performed a chemical screen to investigate WNT signaling in epithelial differentiation. We identify an association between Casein Kinase activity and maintenance of an AT2 expression signature; Casein Kinase inhibition leads to an increase in AT1/progenitor cell ratio. These organoids provide a simplified model of alveolar differentiation and constitute a scalable screening platform to identify and analyze cell differentiation mechanisms.


Assuntos
Diferenciação Celular , Alvéolos Pulmonares/citologia , Células-Tronco/citologia , Animais , Caseína Quinases/antagonistas & inibidores , Caseína Quinases/metabolismo , Células Cultivadas , Colágeno Tipo I/metabolismo , Meios de Cultura Livres de Soro , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Marcadores Genéticos , Camundongos , Camundongos Endogâmicos C57BL , Alvéolos Pulmonares/embriologia , Alvéolos Pulmonares/enzimologia , Alvéolos Pulmonares/metabolismo , Transcrição Genética , Via de Sinalização Wnt
3.
Indian J Ophthalmol ; 69(9): 2452-2456, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34427243

RESUMO

Purpose: To compare the physical and microbiological characteristics of McCarey-Kaufman (MK), Cornisol, and Optisol-GS media and evaluate the outcomes of keratoplasty performed using corneas stored in these three media. Methods: The study involved 60 donor corneas which were distributed in 3 groups: MK, Cornisol, and Optisol-GS. Corneas in these groups were further analyzed based on the type of keratoplasty performed (full thickness versus endothelial keratoplasty). At baseline, the endothelial cell density and death to preservation time of donor corneas were recorded. Following keratoplasty, patients were evaluated on day 1, at 1 month, 3 months, and 6 months follow-up. Outcomes were assessed in terms of corrected distance visual acuity (CDVA), endothelial cell density, percentage endothelial cell loss, and corneal thickness. The storage media were also assessed for their physical quality and their microbiological characteristics. Results: Physical characteristics of all three media were found to be within normal limits. Mean CDVA was comparable among the 3 groups at 6-month follow-up. The absolute endothelial cell count values were significantly lower for corneas stored in MK medium (1873.7 ± 261.1 cells/mm2) compared to the Cornisol (2085.0 ± 230.3 cells/mm2) and Optisol-GS media [(2180.3 ± 217.2 cells/mm2) (P = <0.001)]. Corneas stored in Optisol-GS medium were significantly thinner at 1-month follow-up with no significant difference at 6 months (P = 0.66). Conclusion: Optisol-GS and Cornisol media were found to preserve endothelial cell density better and stabilize corneal thickness earlier as compared to the MK medium. However, the functional outcomes were comparable among the three groups.


Assuntos
Endotélio Corneano , Preservação de Órgãos , Córnea/cirurgia , Meios de Cultura Livres de Soro , Humanos , Doadores de Tecidos
4.
Int J Mol Sci ; 22(12)2021 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-34203724

RESUMO

Numerous studies have shown that hedgehog inhibitors (iHHs) only partially block the growth of tumor cells, especially in vivo. Leukemia often expands in a nutrient-depleted environment (bone marrow and thymus). In order to identify putative signaling pathways implicated in the adaptive response to metabolically adverse conditions, we executed quantitative phospho-proteomics in T-cell acute lymphoblastic leukemia (T-ALL) cells subjected to nutrient-depleted conditions (serum starvation). We found important modulations of peptides phosphorylated by critical signaling pathways including casein kinase, mammalian target of rapamycin, and 5'AMP-activated kinase (AMPK). Surprisingly, in T-ALL cells, AMPK signaling was the most consistently downregulated pathway under serum-depleted conditions, and this coincided with increased GLI1 expression and sensitivity to iHHs, especially the GLI1/2 inhibitor GANT-61. Increased sensitivity to GANT-61 was also found following genetic inactivation of the catalytic subunit of AMPK (AMPKα1) or pharmacological inhibition of AMPK by Compound C. Additionally, patient-derived xenografts showing high GLI1 expression lacked activated AMPK, suggesting an important role for this signaling pathway in regulating GLI1 protein levels. Further, joint targeting of HH and AMPK signaling pathways in T-ALL cells by GANT-61 and Compound C significantly increased the therapeutic response. Our results suggest that metabolic adaptation that occurs under nutrient starvation in T-ALL cells increases responsiveness to HH pathway inhibitors through an AMPK-dependent mechanism and that joint therapeutic targeting of AMPK signaling and HH signaling could represent a valid therapeutic strategy in rapidly expanding tumors where nutrient availability becomes limiting.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Proteínas Hedgehog/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Transdução de Sinais , Proteínas Quinases Ativadas por AMP/genética , Morte Celular/efeitos dos fármacos , Meios de Cultura Livres de Soro/farmacologia , Ativação Enzimática/efeitos dos fármacos , Humanos , Células Jurkat , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Piridinas/farmacologia , Pirimidinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Proteína GLI1 em Dedos de Zinco/metabolismo
5.
Theriogenology ; 172: 47-54, 2021 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-34098168

RESUMO

Protein-free media are essential for the sanitary cryopreservation of bovine genetic resources. Our aim was to set up an optimized protocol for the vitrification of immature bovine oocytes using protein free media which can provide the highest embryo development rates and embryo quality after subsequent in vitro maturation and fertilization. First, using a protein free NCSU-37 as base medium we compared the efficacy of vitrification on Cryotop device with two different CPA protocols. "Protocol A″ employed a combination of ethylene glycol and propylene glycol as permeating cryoprotectants (pCPA) and equilibration in 4% total pCPA (2% ethylene glycol + 2% propylene glycol). "Protocol B″ employed a combination of ethylene glycol and DMSO and equilibration in 15% total pCPA (7.5% ethylene glycol + 7.5% DMSO). The 2 protocols were equally effective in terms of oocyte survival and subsequent development to the blastocyst stage. However, blastocyst cell numbers were significantly higher with "Protocol A". TCM-199 and NCSU-37 were equally effective as base media for vitrification. Vitrification with "Protocol A″ reduced the percentage of live oocytes and subsequent development to blastocyst stage but did not affect the hatching and cell numbers of blastocysts when compared to the non-treated group. CPA treatment of "Protocol A″ without cooling did not affect embryo development. Storage of ovaries in PBS at 15 °C for overnight reduced the percentage of surviving oocytes after vitrification but not their subsequent development to the blastocyst stage. In conclusion we established a vitrification protocol for the cryopreservation of immature bovine oocytes employing protein-free media which provided high blastocyst quality without noticeable toxic effects.


Assuntos
Ovário , Vitrificação , Animais , Blastocisto , Bovinos , Protocolos Clínicos , Criopreservação/veterinária , Crioprotetores/farmacologia , Meios de Cultura Livres de Soro , Etilenoglicol/farmacologia , Feminino , Fertilização In Vitro/veterinária , Oócitos
6.
Sci Rep ; 11(1): 13159, 2021 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-34162924

RESUMO

Nonalcoholic fatty liver disease (NAFLD) is the most common liver disease and strongly correlates with the growing incidence of obesity and type II diabetes. We have developed a human-on-a-chip model composed of human hepatocytes and adipose tissue chambers capable of modeling the metabolic factors that contribute to liver disease development and progression, and evaluation of the therapeutic metformin. This model uses a serum-free, recirculating medium tailored to represent different human metabolic conditions over a 14-day period. The system validated the indirect influence of adipocyte physiology on hepatocytes that modeled important aspects of NAFLD progression, including insulin resistant biomarkers, differential adipokine signaling in different media and increased TNF-α-induced steatosis observed only in the two-tissue model. This model provides a simple but unique platform to evaluate aspects of an individual factor's contribution to NAFLD development and mechanisms as well as evaluate preclinical drug efficacy and reassess human dosing regimens.


Assuntos
Adipócitos/efeitos dos fármacos , Descoberta de Drogas/instrumentação , Hepatócitos/efeitos dos fármacos , Hipoglicemiantes/farmacologia , Dispositivos Lab-On-A-Chip , Metformina/farmacologia , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Adipócitos/metabolismo , Tecido Adiposo Branco/citologia , Comunicação Celular , Células Cultivadas , Meios de Cultura/farmacologia , Meios de Cultura Livres de Soro/farmacologia , Citocromo P-450 CYP1A1/metabolismo , Citocromo P-450 CYP3A/metabolismo , Desenho de Equipamento , Ácidos Graxos/metabolismo , Ácidos Graxos/farmacologia , Glucose/farmacologia , Hepatócitos/metabolismo , Humanos , Inflamação , Insulina/farmacologia , Fator de Necrose Tumoral alfa/farmacologia
7.
Sci Rep ; 11(1): 12233, 2021 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-34112824

RESUMO

Type 2 innate lymphoid cells (ILC2s) were discovered approximately ten years ago and their clinical relevance is gaining greater importance. However, their successful isolation from mammalian tissues and in vitro culture and expansion continues to pose challenges. This is partly due to their scarcity compared to other leukocyte populations, but also because our current knowledge of ILC2 biology is incomplete. This study is focused on ST2+ IL-25Rlo lung resident ILC2s and demonstrate for the first time a methodology allowing mouse type 2 innate lymphoid cells to be cultured, and their numbers expanded in serum-free medium supplemented with Interleukins IL-33, IL-2, IL-7 and TSLP. The procedures described methods to isolate ILC2s and support their growth for up to a week while maintaining their phenotype. During this time, they significantly expand from low to high cell concentrations. Furthermore, for the first time, sub-cultures of primary ILC2 purifications in larger 24- and 6-well plates were undertaken in order to compare their growth in other media. In culture, ILC2s had doubling times of 21 h, a growth rate of 0.032 h-1 and could be sub-cultured in early or late phases of exponential growth. These studies form the basis for expanding ILC2 populations that will facilitate the study and potential applications of these rare cells under defined, serum-free conditions.


Assuntos
Técnicas de Cultura de Células , Meios de Cultura Livres de Soro , Imunidade Inata , Subpopulações de Linfócitos/imunologia , Subpopulações de Linfócitos/metabolismo , Animais , Biomarcadores , Células Cultivadas , Citocinas/biossíntese , Subpopulações de Linfócitos/citologia , Camundongos
8.
Biomaterials ; 275: 120842, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34087583

RESUMO

Defective cellular metabolism, impaired mitochondrial function, and increased cell death are major problems that adversely affect donor tissues during hypothermic preservation prior to transplantation. These problems are thought to arise from accumulated reactive oxygen species (ROS) inside cells. Oxidative stress acting on the cells of organs and tissues preserved in hypothermic conditions before surgery, as is the case for cornea transplantation, is thought to be a major reason behind cell death prior to surgery and decreased graft survival after transplantation. We have recently discovered that ubiquinol - the reduced and active form of coenzyme Q10 and a powerful antioxidant - significantly enhances mitochondrial function and reduces apoptosis in human donor corneal endothelial cells. However, ubiquinol is highly lipophilic, underscoring the need for an aqueous-based formulation of this molecule. Herein, we report a highly dispersible and stable formulation comprising a complex of ubiquinol and gamma cyclodextrin (γ-CD) for use in aqueous-phase ophthalmic products. Docking studies showed that γ-CD has the strongest binding affinity with ubiquinol compared to α- or ß-CD. Complexed ubiquinol showed significantly higher stability compared to free ubiquinol in different aqueous ophthalmic products including Optisol-GS® corneal storage medium, balanced salt solution for intraocular irrigation, and topical Refresh® artificial tear eye drops. Greater ROS scavenging activity was noted in a cell model with high basal metabolism and ROS generation (A549) and in HCEC-B4G12 human corneal endothelial cells after treatment with ubiquinol/γ-CD compared to free ubiquinol. Furthermore, complexed ubiquinol was more effective at lowering ROS, and at far lower concentrations, compared to free ubiquinol. Complexed ubiquinol inhibited lipid peroxidation and protected HCEC-B4G12 cells against erastin-induced ferroptosis. No evidence of cellular toxicity was detected in HCEC-B4G12 cells after treatment with complexed ubiquinol. Using a vertical diffusion system, a topically applied inclusion complex of γ-CD and a lipophilic dye (coumarin-6) demonstrated transcorneal penetrance in porcine corneas and the capacity for the γ-CD vehicle to deliver drug to the corneal endothelium. Using the same model, topically applied ubiquinol/γ-CD complex penetrated the entire thickness of human donor corneas with markedly greater ubiquinol retention in the endothelium compared to free ubiquinol. Lastly, the penetrance of ubiquinol/γ-CD complex was assayed using human donor corneas preserved for 7 days in Optisol-GS® per standard industry practices, and demonstrated higher amounts of ubiquinol retained in the corneal endothelium compared to free ubiquinol. In summary, ubiquinol complexed with γ-CD is a highly stable composition that can be incorporated into a variety of aqueous-phase products for ophthalmic use including donor corneal storage media and topical eye drops to scavenge ROS and protect corneal endothelial cells against oxidative damage.


Assuntos
Transplante de Córnea , Células Endoteliais , Animais , Córnea , Meios de Cultura Livres de Soro , Dextranos , Endotélio Corneano , Gentamicinas , Humanos , Preservação de Órgãos , Suínos , Ubiquinona/análogos & derivados
9.
Int J Mol Sci ; 22(9)2021 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-34066474

RESUMO

Corneal endothelial dystrophy is a relevant cause of vision loss and corneal transplantation worldwide. In the present study, we analyzed the effect of mesenchymal stem cell (MSC)-derived extracellular vesicles (MSC-EVs) in an in vitro model of corneal dystrophy, characterized by endoplasmic reticulum stress. The effects of MSC-EVs were compared with those of serum-derived EVs, reported to display a pro-angiogenic activity. MSC-EVs were able to induce a significant down-regulation of the large majority of endoplasmic reticulum stress-related genes in human corneal endothelial cells after exposure to serum deprivation and tunicamycin. In parallel, they upregulated the Akt pathway and limited caspase-3 activation and apoptosis. At variance, the effect of the serum EVs was mainly limited to Akt phosphorylation, with minimal or absent effects on endoplasmic reticulum stress modulation and apoptosis prevention. The effects of MSC-EVs were correlated to the transfer of numerous endoplasmic reticulum (ER)-stress targeting miRNAs to corneal endothelial cells. These data suggest a potential therapeutic effect of MSC-EVs for corneal endothelial endoplasmic reticulum stress, a major player in corneal endothelial dystrophy.


Assuntos
Apoptose , Estresse do Retículo Endoplasmático , Células Endoteliais/patologia , Endotélio Corneano/patologia , Vesículas Extracelulares/metabolismo , Células-Tronco Mesenquimais/metabolismo , Apoptose/efeitos dos fármacos , Apoptose/genética , Separação Celular , Meios de Cultura Livres de Soro , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Estresse do Retículo Endoplasmático/genética , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Vesículas Extracelulares/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Células-Tronco Mesenquimais/efeitos dos fármacos , MicroRNAs/genética , MicroRNAs/metabolismo , Fosforilação/efeitos dos fármacos , Tunicamicina/farmacologia
10.
Cell Tissue Bank ; 22(4): 563-574, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-33937957

RESUMO

To compare ex vivo results of donor corneas maintained in Sinasol with those stored in Optisol-GS and reporting clinical outcomes of grafted Sinasol-versus Optisol-GS-stored corneas. In phase I, paired donor corneas were maintained in Sinasol or Optisol-GS. Afterward, the corneas were subjected to slit-lamp biomicroscopic and specular microscopic examinations on days 1 and 7, and then to trypan blue staining on day 7. The same examinations were performed on the corneas that were kept in Sinasol or Optisol-GS for 14 days. In phase II, the post-operative reports of 72 consecutive corneal transplantations were recorded using Sinasol- or Optisol-GS-preserved corneas. In phase I, 128 corneas from 64 donors and 59 corneas from 33 donors were investigated for 7 and 14 days, respectively. The EC indices were comparable between the groups at the measurement periods. The EC losses over 7 and 14 days were 3.7% and 19.9% in Sinasol against 4.6% and 20.8% in Optisol-GS. Although fair quality corneas were more common in Optisol-GS group after 7 (P = 0.04) and 14 days (P = 0.034), changes of stromal edema, Descemet's fold, and other quality ratings during 14 days were not different between the groups. In phase II, all the transplanted corneas were postoperatively clear with no adverse reactions. The overall results indicate that Sinasol is a safe, effective, and affordable intermediate cold storage medium for preservation of corneas.


Assuntos
Soluções para Preservação de Órgãos , Sulfatos de Condroitina , Misturas Complexas , Córnea/cirurgia , Meios de Cultura Livres de Soro , Dextranos , Endotélio Corneano , Gentamicinas , Humanos , Preservação de Órgãos , Estudos Prospectivos
11.
Tissue Eng Regen Med ; 18(3): 355-367, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-34047999

RESUMO

BACKGROUND: In order to produce and isolate the exosome derived from the cell of interests, a serum free environment (starvation) has been essential for excluding the unknown effect from serum-derived exosomes. Recently, serum-free culture media have been developed as a substitute for serum supplemented media so that MSC proliferates with maintaining the original characteristics of the cells in a serum free condition. Due to the different properties of the exosomes representing the states and characteristics of the origin cells, a study is needed to compare the properties of the cell-derived exosomes according to the cell culture media. METHODS: To compare the cell culture condition on exosomes, human umbilical cord mesenchymal stem cells (UCMSCs) were cultured with two different media, serum containing media, 10% FBS supplemented DMEM (NM) and serum-free chemically defined media, CellCor™ CD MSC (CDM). To remove FBS-derived exosomes from UCMSC cultured with NM, the medium was replaced with FBS-free DMEM for starvation during exosome isolation. The production yield and expression levels of angiogenic and pro-inflammatory factors were compared. And, the subpopulations of exosome were classified depending on the surface properties and loaded cytokines. Finally, the wound healing and angiogenic effects have been evaluated using in vitro assays. RESULTS: The UCMSC-derived exosomes under two different cell culture media could be classified into subpopulations according to the surface composition and loaded cytokines. Especially, exosome derived from UCMSC cultured with CDM showed higher expression levels of cytokines related to regenerative bioactivities which resulted in enhanced wound healing and angiogenesis. CONCLUSION: CDM has the advantages to maintain cell proliferation even during the period of exosome isolations and eliminate unknown side effects caused by serum-derived exosomes. Additionally, exosomes derived from UCMSC cultured with CDM show better wound healing and angiogenic effects due to a lot of regeneration-related cytokines and less pro-inflammatory cytokines compared to with NM.


Assuntos
Exossomos , Células-Tronco Mesenquimais , Técnicas de Cultura de Células , Meios de Cultura Livres de Soro , Humanos , Cordão Umbilical
12.
Biotechnol Bioeng ; 118(7): 2649-2659, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33837958

RESUMO

The Vero cell line is the most used continuous cell line in viral vaccine manufacturing. This adherent cell culture platform requires the use of surfaces to support cell growth, typically roller bottles, or microcarriers. We have recently compared the production of rVSV-ZEBOV on Vero cells between microcarrier and fixed-bed bioreactors. However, suspension cultures are considered superior with regard to process scalability. Therefore, we further explore the Vero suspension system for recombinant vesicular stomatitis virus (rVSV)-vectored vaccine production. Previously, this suspension cell line was only able to be cultivated in a proprietary medium. Here, we expand the adaptation and bioreactor cultivation to a serum-free commercial medium. Following small-scale optimization and screening studies, we demonstrate bioreactor productions of highly relevant vaccines and vaccine candidates against Ebola virus disease, HIV, and coronavirus disease 2019 in the Vero suspension system. rVSV-ZEBOV, rVSV-HIV, and rVSVInd -msp-SF -Gtc can replicate to high titers in the bioreactor, reaching 3.87 × 107 TCID50 /ml, 2.12 × 107 TCID50 /ml, and 3.59 × 109 TCID50 /ml, respectively. Furthermore, we compare cell-specific productivities, and the quality of the produced viruses by determining the ratio of total viral particles to infectious viral particles.


Assuntos
Reatores Biológicos/virologia , Técnicas de Cultura de Células/métodos , Vacinas contra Ebola , Vesiculovirus/genética , Animais , Vacinas contra COVID-19 , Chlorocebus aethiops , Meios de Cultura Livres de Soro , Células Vero , Vacinas Virais
13.
Cells ; 10(4)2021 04 13.
Artigo em Inglês | MEDLINE | ID: mdl-33924517

RESUMO

Mesenchymal stem cells (MSCs) are of great interest for their use in cell-based therapies due to their multipotent differentiation and immunomodulatory capacities. In consequence of limited numbers following their isolation from the donor tissue, MSCs require extensive expansion performed in traditional 2D cell culture setups to reach adequate amounts for therapeutic use. However, prolonged culture of MSCs in vitro has been shown to decrease their differentiation potential and alter their immunomodulatory properties. For that reason, preservation of these physiological characteristics of MSCs throughout their in vitro culture is essential for improving the efficiency of therapeutic and in vitro modeling applications. With this objective in mind, many studies already investigated certain parameters for enhancing current standard MSC culture protocols with regard to the effects of specific culture media components or culture conditions. Although there is a lot of diversity in the final therapeutic uses of the cells, the primary stage of standard isolation and expansion is imperative. Therefore, we want to review on approaches for optimizing standard MSC culture protocols during this essential primary step of in vitro expansion. The reviewed studies investigate and suggest improvements focused on culture media components (amino acids, ascorbic acid, glucose level, growth factors, lipids, platelet lysate, trace elements, serum, and xenogeneic components) as well as culture conditions and processes (hypoxia, cell seeding, and dissociation during passaging), in order to preserve the MSC phenotype and functionality during the primary phase of in vitro culture.


Assuntos
Técnicas de Cultura de Células , Separação Celular/métodos , Meios de Cultura Livres de Soro/farmacologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Aminoácidos/química , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Terapia Baseada em Transplante de Células e Tecidos/métodos , Células Cultivadas , Meios de Cultura Livres de Soro/química , Humanos , Imunomodulação , Peptídeos e Proteínas de Sinalização Intercelular/química , Lipídeos/química , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/imunologia , Oligoelementos/química
14.
Sci Rep ; 11(1): 8096, 2021 04 14.
Artigo em Inglês | MEDLINE | ID: mdl-33854099

RESUMO

Stroke causes death and disability globally but no neuroprotectant is approved for post-stroke neuronal injury. Neuroprotective compounds can be identified using oxygen glucose deprivation (OGD) of neuronal cells as an in vitro stroke model. Nerve growth factor (NGF)-differentiated PC12 pheochromocytoma cells are frequently used. However, investigators often find their clonal variant undifferentiable and are uncertain of optimal culture conditions. Hence we studied 3 commonly used PC12 variants: PC12 Adh, PC12 from Riken Cell Bank (PC12 Riken) and Neuroscreen-1 (NS-1) cells. We found DMEM the optimal media for PC12 Riken and NS-1 cells. Using a novel serum-free media approach, we identified collagen IV as the preferred adhesive substrate for both cell lines. We found PC12 Adh cells cannot attach without serum and is unable to differentiate using NGF. NS-1 cells differentiated to a maximal 72.7 ± 5.2% %, with substantial basal differentiation. We optimised differentiated NS-1 cells for an in vitro stroke model using 3 h of OGD resulting in ~ 70% viable cells. We screened 5 reported neuroprotectants and provide the first report that serotonin is antiapoptotic in a stroke model and the 5-HT1A agonist 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT) is neuroprotective in PC12 cells. Thus we demonstrate the optimisation and validation for a PC12 cell-based in vitro stroke model.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Modelos Biológicos , Fármacos Neuroprotetores/farmacologia , 8-Hidroxi-2-(di-n-propilamino)tetralina/farmacologia , Animais , Apoptose/efeitos dos fármacos , Hipóxia Celular , Sobrevivência Celular/efeitos dos fármacos , Meios de Cultura Livres de Soro/química , Fármacos Neuroprotetores/uso terapêutico , Células PC12 , Ratos , Serotonina/farmacologia , Agonistas do Receptor 5-HT1 de Serotonina/farmacologia , Acidente Vascular Cerebral/tratamento farmacológico , Acidente Vascular Cerebral/patologia
15.
Hum Cell ; 34(3): 819-824, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33677815

RESUMO

Mesenchymal stem cells (MSCs) have recently made significant progression in multiple clinical trials targeting several clinical disorders and in the modulation of immune responses. In the present study, we isolated human adipose tissue-derived stem cells (ADSCs) by direct membrane migration method without using enzymatic digestion via collagenase, and tried to extract adequate number of cells for clinical application. Hydroxyapatite-treated nonwoven fabric membrane made up of synthetic macromolecular fiber materials, polyethylene and polyester terephthalene was used. Expansion culture of ADSCs having plastic flask adherent characteristic in serum-free condition was successfully established, and adequate number of cells were obtained for clinical application. They were found to be positive for CD44, CD73, CD90 and CD105 and negative for CD11b, CD34, CD45, CD80 and HLA-DR. The resulting immunological marker profile satisfied the immunophenotype of previously reported MSCs. Also, microscopic findings demonstrated trilineage differentiation into adipogenic, osteogenic and chondrogenic cells as the characteristics of MSCs. The isolation by nonwoven fabric membrane and expanded cells under serum-free condition satisfied the criteria of MSCs, as proposed by the International Society for Cellular Therapy. Our direct membrane migration method without enzyme digestion is useful as ADSCs can be obtained from small pieces of adipose tissue and expanded under serum-free culture condition. This method was considered to be feasible for clinical application.


Assuntos
Tecido Adiposo/citologia , Separação Celular/métodos , Membranas Artificiais , Células-Tronco Mesenquimais , Antígenos CD/metabolismo , Células Cultivadas , Meios de Cultura Livres de Soro , Durapatita , Humanos , Células-Tronco Mesenquimais/metabolismo , Poliésteres , Polietileno
17.
In Vitro Cell Dev Biol Anim ; 57(3): 300-314, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33537930

RESUMO

Cancer metastasis and recurrence are potentially lethal. A small number of cancer cell groups called cancer stem cells (CSCs) have both stem cell capacity and cancer-forming ability and are reported to play important roles in cancer metastasis and recurrence. These CSCs are considered to be radiation-resistant (RR). Therefore, understanding the biological effects of radiation on squamous cell carcinoma (SCC) cell lines in vitro and in vivo might be worthwhile to circumvent radiation resistance. Currently, there are no reports on the establishment of RR-SCC cells in serum-free defined culture, which mimics biological mechanisms and prevents instability of using serum in the culture medium. We isolated radiation-resistant strains, designated A431-LDR and A431-HDR, from A431 cells derived from vulval SCC and irradiated them with a total dose of 60 Gy at a low-dose rate (2.2 Gy/d) (RM1000) and a high-dose rate (5 Gy/5.75min) in serum-free defined culture. These cells exhibited high sphere-forming and migration ability in vitro and high tumor-forming ability in nude mice xenografts. Overexpression of KRT13 in A431-RR cells might play a role in its radiation-resistant characteristics. These cells might be useful not only to study cancer stem cells but also to study the circumvention of radiation resistance by novel cancer treatment modalities.


Assuntos
Carcinoma de Células Escamosas/genética , Regulação Neoplásica da Expressão Gênica , Queratina-13/genética , Tolerância a Radiação/genética , Neoplasias Vulvares/genética , Animais , Carcinogênese/patologia , Carcinogênese/efeitos da radiação , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Movimento Celular/efeitos da radiação , Proliferação de Células/efeitos da radiação , Meios de Cultura Livres de Soro , Relação Dose-Resposta à Radiação , Feminino , Humanos , Queratina-13/metabolismo , Camundongos Nus , Células-Tronco Neoplásicas/patologia , Células-Tronco Neoplásicas/efeitos da radiação , RNA Interferente Pequeno/metabolismo , Ensaio Tumoral de Célula-Tronco , Neoplasias Vulvares/patologia
18.
Sci Rep ; 11(1): 3403, 2021 02 09.
Artigo em Inglês | MEDLINE | ID: mdl-33564114

RESUMO

Bone marrow-derived mesenchymal stromal cells (BM-MSCs) are gaining increasing importance in the field of regenerative medicine. Although therapeutic value of MSCs is now being established through many clinical trials, issues have been raised regarding their expansion as per regulatory guidelines. Fetal bovine serum usage in cell therapy poses difficulties due to its less-defined, highly variable composition and safety issues. Hence, there is a need for transition from serum-based to serum-free media (SFM). Since SFM are cell type-specific, a precise analysis of the properties of MSCs cultured in SFM is required to determine the most suitable one. Six different commercially available low serum/SFM with two different seeding densities were evaluated to explore their ability to support the growth and expansion of BM-MSCs and assess the characteristics of BM-MSCs cultured in these media. Except for one of the SFM, all other media tested supported the growth of BM-MSCs at a low seeding density. No significant differences were observed in the expression of MSC specific markers among the various media tested. In contrary, the population doubling time, cell yield, potency, colony-forming ability, differentiation potential, and immunosuppressive properties of MSCs varied with one another. We show that SFM tested supports the growth and expansion of BM-MSCs even at low seeding density and may serve as possible replacement for animal-derived serum.


Assuntos
Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Técnicas de Cultura de Células , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Adolescente , Adulto , Meios de Cultura Livres de Soro/farmacologia , Feminino , Humanos , Masculino
19.
Methods Mol Biol ; 2286: 95-105, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33534112

RESUMO

Bone marrow stromal cells (BMSCs, also known as bone marrow mesenchymal stem cells) are a plastic-adherent heterogeneous cell population that contain inherent skeletal progenitors and a subset of multipotential skeletal stem cells (SSCs). Application of BMSCs in therapeutic protocols implies its isolation and expansion under good manufacturing practices (GMP). Here we describe the procedures we have found to successfully generate practical BMSCs numbers, with preserved biological potency.


Assuntos
Tecnologia Biomédica/normas , Células da Medula Óssea/citologia , Osso e Ossos/citologia , Cultura Primária de Células/métodos , Antígenos CD34/genética , Antígenos CD34/metabolismo , Tecnologia Biomédica/métodos , Células Cultivadas , Técnicas de Cocultura/economia , Técnicas de Cocultura/métodos , Técnicas de Cocultura/normas , Custos e Análise de Custo , Meios de Cultura Livres de Soro/química , Humanos , Guias de Prática Clínica como Assunto , Cultura Primária de Células/economia , Cultura Primária de Células/normas , Células Estromais/citologia , Células Estromais/metabolismo
20.
Arq Bras Oftalmol ; 84(2): 163-169, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33567016

RESUMO

PURPOSE: The aim of this study was to evaluate the physical and chemical characteristics of coconut water and to analyze the use of coconut water solution for the conservation of human corneas. METHODS: This was an experimental and controlled study performed at the Eye Bank of the General Hospital of Fortaleza. The coconut water-based solution was prepared at the Goat Seed Technology Laboratory of the Department of Veterinary Medicine of the State University of Ceará. Discarded corneas from the Eye Bank were divided into two groups for sequential experiments: G1, coconut water-based solution (experimental group), and G2, conservative treatment with OPTISOL GS® (control group). The osmolality of corneas in G1 was analyzed sequentially at 275, 300, 325, 345, 365, and 400 mOsm/L. The viability of the corneas was determined by specular microscopy and biomicroscopy on the first, third, and seventh days. RESULTS: Corneas preserved in a solution of 365 and 345 mOsm/L had a transparency of 8 mm until the third day and had diffuse edema in the periphery, central folds, and partial epithelium loss until the seventh day. The 365-mOsm/L solution was associated with the worst results during follow-up. Corneas placed in Optisol-GS retained their original aspects. CONCLUSIONS: Coconut water-based preservative partially maintained corneal transparency and epithelial integrity, especially during the first three days of follow-up. The coconut water-based solutions used were not effective for use as preservatives in a human eye bank.


Assuntos
Cocos , Preservação de Órgãos , Córnea , Meios de Cultura Livres de Soro , Humanos , Água
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