Effects of different storage conditions on the content of key growth factors in platelet-rich plasma / 中国输血杂志

【Objective】 To study the effects of different storage temperature and different storage time on the activity of key growth factors in platelet-rich plasma(PRP), and to provide a theoretical basis for maximize the role of PRP in clinical treatment. 【Methods】 PRP was collected by blood cell isolation and apheresis, stored at 22℃ and -80℃, respectively. VEGF, TGF-β and PDGF were detected by ELISA. The content of growth factors in PRP was detected when stored at 22℃for 1, 3 and 5 days, and the growth factors content of PRP stored at 22℃ for 3 days was detected after thrombin activation for 0.5, 1 and 1.5 hours. The content of growth factor in frozen PRP (stored at -80℃ for 30 days after initial 3-days storage at 22℃ ) and fresh PRP (stored at 22℃ for 3 days) was compared. The growth factor content in PRP frozen at - 80℃ for 30, 60 and 180 days, and the growth factor content in PRP frozen at -80℃ for 180 days after repeated freeze-thaw for 1, 2, 3, 5 and 10 times were detected. 【Results】 The growth factor content of apheresis PRP was significantly higher than that of platelet-poor plasma. No statistical difference was noticed in VEGF, TGF-β and PDGF content in PRP at 1, 3 and 5 days stored at 22℃; no statistical difference was noticed in VEGF, TGF-β and PDGF content in PRP stored at 22℃ for 3 days after thrombin activation for 0.5, 1 and 1.5 hours. There was no statistically significant difference in growth factor content between PRP stored at 22℃ for 3 days versus frozen at -80℃ for 30 days after initial 3-days storage at 22℃. No statistical difference was found in VEGF, TGF-β and PDGF contents in frozen PRP repeatedly frozen and thawed for 1 to 10 times. 【Conclusion】 Apheresis PRP can release a large amount of growth factors after activation. Fresh PRP stored at 22℃ for 5 days or frozen at -80℃ for 180 days has no impact on the content of growth factors, and frozen PRP at -80℃ can achieve long-term, effective and safe preservation, which is conducive to multiple use of PRP in treatment.

Ammonium chloride lysis method versusnon-lysis method for isolation of human adipose-derived stem cells / 中国组织工程研究

BACKGROUND: A general standard has not been established for the extraction and purification of adipose-derived stem cells (ADSCs). An erythrocyte lysis step for stromal vascular fraction is the commonly used method in the procedure for ADSCs isolation. However, there is a lack of evidence on whether this step will have adverse effects on human ADSCs (hADSCs). OBJECTIVE: To test the efficiency of two hADSCs isolation methods, which are erythrocyte-lysis method based on ammonium chloride and non-lysis method. Moreover, the biological characteristics of the hADSCs isolated by the two methods were also compared. METHODS: After collagenase digestion of lipoaspirate, erythrocyte lysis buffer was used to purify stromal vascular fraction in erythrocyte-lysis method, while in non-lysis method the buffer was not used. A Muse™ cell analyzer was used to assess living cell counting and proportion of stromal vascular fraction in both methods. Then hADSCs were cultured to the second passage for next testing. Cell morphology was observed under light microscope. Cell phenotype was detected by flow cytometry. Cell counting kit-8 was used to evaluate cell proliferation. Oil red O staining and alizarin staining were used to evaluate adipogenic and osteogenic ability of hADSCs after adipogenic and osteogenic induction. This study was approved by the Ethics Committee of the Plastic Surgery Hospital, Peking Union Medical College, Chinese Academy of Medical Sciences, and informed consents were signed by all participants. RESULTS AND CONCLUSION: (1) Compared with the erythrocyte lysis group, hADSCs obtained in the non-lysis group contained a larger number and a larger percentage of non-erythrocyte living cells. (2) The two groups of hADSCs were spindle-shaped and arranged as a fish shape. (3) The cell phenotypes of both groups met the phenotypic requirements for human mesenchymal stem cells. (4) The cell proliferation in the non-lysis group was faster than that in the erythrocyte lysis group, while there was no difference in the adipogenic and osteogenic abilities between the two groups. In conclusion, the use of erythrocyte lysis buffer reduces the isolation efficiency of hADSCs and inhibits cell proliferation. The non-lysis isolation method does not affect phenotypes, adipogenic and osteogenic ability of hADSCs. Therefore, it is not recommended to implement erythrocyte lysis during the isolation of hADSCs.

Isolation, identification and cultivation of primary rat hepatic stellate cells / 局解手术学杂志

Objeetive To improve the method for the isolation and purification of rat hepatic stellate(HSC) cells and to provide a stable cell source for the research on liver-related diseases.Methods Rat liver was digested in situ by a two-step infusion assay under a strict control of the infusion temperature,flow rate and time with a combined utilization of Pronase E and Collagenase Ⅳ.And then,the HSC cells were separated by Percoll density gradient centrifugation.The cell growth curve and survival rate were measured by CCK-8 and trypan blue staining,respectively.The HSC cells were identified by flow cytometry and immunofluorescence cytochemistry.Results With the improved methods,there were (2.1 ± 0.2) × 107 HSC cells isolated from one rat and the survival rate was (96.2 ± 0.8) ％.The percentage of HSC cells with a spontaneous fluorescent characteristic from the isolated cells was 96.3％.The immunofluorescence cytochemistry was used to detect the expressions of the surface antigens α-SMA and Desmin in the isolated HSC cells.Conclusion By strict control of infusion temperature,flow rate and perfusion time as well as the combined application of Pronase E and Collagenase Ⅳ,there is an increased harvest of HSC cells with improved cell viability and purity,which is helpful for further research on HSC cells.

Comparison of the culture methods between human cumulus cells and mural granulosa cells / 中国病理生理杂志

AIM:To establish an effective method for purification and culture of human cumulus cells(CCs) in vitro,and to compare the characteristics between CCs and mural granulosa cells(MGCs).METHODS:Follicular fluid and cumulus complex from the patients undergoing intracytoplasmic sperm injection were collected.CCs were mechanically cut from cumulus complex and then directly inoculated on a Petri dish, and MGCs were obtained from follicular fluid through density gradient centrifugation.The expression of follicle stimulating hormone receptor(FSHR)was determined by immunofluorescence.The cell growth curves were measured by CCK-8 assay.The secretion of estrogen was detected by ELISA.RESULTS:After incubated for 24 h, the adherence of CCs was observed.CCs and MGCs had similar growth characteristics and FSHR expression.The similar cell growth curves were observed by CCK-8 assay and the results of ELISA showed that they had comparable secretion of estrogen.CONCLUSION:Direct culture of CCs mechanically cutting from cumulus complex is an effective method.CCs had similar growth characteristics,growth curves and secretion of estro-gen to MGCs cultured in vitro and could be a substitutive source of granulosa cell subsets.

Comparação de dois métodos de colheita de medula óssea de equinos e de dois meios de diluição para o isolamento de células mononucleares / Comparison of two methods of equine bone marrow aspiration and two solutions for mononuclear cell isolation

O objetivo do presente estudo foi avaliar a concentração e viabilidade da fração de células mononucleares (FCM) a partir de diferentes técnicas de colheita e processamento de medula óssea (MO) em equinos. Foram avaliados cinco equinos adultos, hígidos e sem raça definida. Obtiveram-se frações de medula óssea (MO) do osso esterno, de acordo com dois protocolos: na colheita A, utilizou-se 10mL de solução de heparina dentro da seringa e em seguida, aspirou-se a MO; na colheita B, 10mL de solução de heparina foi injetada na MO e a aspiração foi realizada após 20 segundos. Todos os animais foram submetidos aos dois protocolos de colheitas, realizadas em sequência, sem intervalo entre os dois procedimentos. Após isolamento da fração de células mononucleares (FCM), das amostras de MO obtidas nas colheitas A e B, cada amostra foi dividida em dois tubos, um contendo solução de DMEM e outro contendo PBS. Assim, alternando-se o tipo de colheita e a solução diluidora, obteve-se quatro tubos de amostras por animal. Os tubos foram centrifugados e os sedimentos foram homogeneizados nos respectivos meios obtendo-se o volume final de 100μL. Realizou-se determinação da concentração e viabilidade celular, obtendo-se as concentrações médias de FCM. Para ambos os meios de diluição, a colheita B apresentou valor numérico maior em comparação à colheita A, porém não foi significativo (p>0,05). Atribui-se tal tendência à menor ocorrência de coagulação da MO no momento da colheita B, sugerindo-se melhor aproveitamento da FCM. Não houve diferença (p>0,05) entre os meios DMEM ou PBS, indicando que os mesmos não alteraram a viabilidade celular. Os protocolos utilizados para colheita de MO e separação da FCM se mostraram eficientes, para o uso em terapia celular em equinos.

The aim of this study was to evaluate mononuclear cells fraction (MCF) concentration and viability from different techniques of bone marrow (BM) aspiration and processing in horses. Five adult horses, healthy and of unknown breed were evaluated. BM was obtained from sternum bone, according two protocols: in aspiration A, 10mL of heparin solution was used inside the syringe and BM was aspirated; in aspiration B, 10mL of heparin solution was injected into the BM, and aspiration was done after 20 seconds. All the animals were submitted by both protocols realized in sequence, without a gap between the procedures. After MCF isolation, of BM samples obtained from A and B aspiration, each sample was divided into two tubes; one contained DMEM solution and the other with PBS solution. Therefore, interchanging the aspiration protocol and the dilution solution, four sample tubes were obtained for each horse. The tubes were centrifuged and the pellet was homogenized with the respectively solution to obtain the final volume of 100μL. Cellular concentration and viability were determined to obtain the FCM medium concentration. For both solutions, the aspiration B had higher numeric values comparing with aspiration A; however, it was not significant (p>0.05). This tendency is attribute for the less BM coagulation observed in the aspiration B, suggesting greater improvement of MCF. No difference (p>0.05) was found between DMEM and PBS solution, indicating that both do not alter the cell viability. The protocols used for BM aspiration and MCF isolation were efficient for application in equine cellular therapy.

Adipose-Derived Stem Cells: An Optimized Protocol for Isolation and Proliferation.

Background and Aims: Current advances in the researches on the stem cells has opened new approaches for their apply in tissue engineering and clinical trials. The most common sources of stem cells are adult and embryonic stem cells. Due to ethical issues, embryonic stem cells use in research has been hotly debated. Unlike embryonic stem cells, adult stem cell have not ethical problem for clinical and research. The purpose of present study was to stem cells isolation and proliferation from the human dipose tissue. Materials and Methods: In this study, stem cells were successfully isolated from human adipose tissue by digestion with type I collagenase enzymes. In summary, adipose tissue were digested by type I collagenase enzyme. Subsequently, the cell solution was centrifuged for mature adipocytes and debris elimination and obtained sedimentation was cultured in culture medium contains Dulbecco's Modified Eagle's Medium (DMEM) via 1% penicillin/streptomycin antibiotics and 10% fetal bovine serum (FBS) at 37°C, 5% CO2 and 95% humidity. Results: in this study, ADSCs were successfully isolated and proliferated. Human ADSCs were able to divide in our culture mediums. Conclusion: The results of the current study were suggesting that this adipose-derived stem cell isolation protocol provides an effective and improved method for isolation and proliferation of these cells in order to tissue engineering application.

Establishment of a primary culture protocol of Mongolian gerbil hepatic stellate cells / 中国比较医学杂志

Objective To investigate the method to isolate and culture hepatic stellate cells ( HSCs) for studying the cellular mechanisms of hepatic frbrosis.Methods HSCs were isolated by nycodenz density gradient centrifugation after the hepatocytes obtained from adult male gebils were digested with pronase, collagenase and DNase, infused via portal vein.The cell viability was determined by trypan blue exclusion test.The purity of HSCs was identified by detectingα-SMA, desmin immunohistochemical staining.Results The yield rate of HSCs was 0.5~1 ×107 per gerbil liver, and the cell viability was more than 90%.The percentage ofα-SMA-positive cells was more than 75%after 3 days primary culture and almost 100% cells were α-SMA and desmin positive in passage culture.Conclusion The successful protocol of primary culture of Mongolian gerbil HSC provide a technical support for research of relevant liver diseases and drug development in the future.

Morphology and morphometry of feline bone marrow-derived mesenchymal stem cells in culture / Morfologia e morfometria das células-tronco mesenquimais isoladas de medula óssea de gato

Mesenchymal stem cells (MSC) are increasingly being proposed as a therapeutic option for treatment of a variety of different diseases in human and veterinary medicine. Stem cells have been isolated from feline bone marrow, however, very few data exist about the morphology of these cells and no data were found about the morphometry of feline bone marrow-derived MSCs (BM-MSCs). The objectives of this study were the isolation, growth evaluation, differentiation potential and characterization of feline BM-MSCs by their morphological and morphometric characteristics. in vitro differentiation assays were conducted to confirm the multipotency of feline MSC, as assessed by their ability to differentiate into three cell lineages (osteoblasts, chondrocytes, and adipocytes). To evaluate morphological and morphometric characteristics the cells are maintained in culture. Cells were observed with light microscope, with association of dyes, and they were measured at 24, 48, 72 and 120h of culture (P1 and P3). The non-parametric ANOVA test for independent samples was performed and the means were compared by Tukey's test. On average, the number of mononuclear cells obtained was 12.29 (±6.05x106) cells/mL of bone marrow. Morphologically, BM-MSCs were long and fusiforms, and squamous with abundant cytoplasm. In the morphometric study of the cells, it was observed a significant increase in average length of cells during the first passage. The cell lengths were 106.97±38.16µm and 177.91±71.61µm, respectively, at first and third passages (24 h). The cell widths were 30.79±16.75 µm and 40.18±20.46µm, respectively, at first and third passages (24 h).The nucleus length of the feline BM-MSCs at P1 increased from 16.28µm (24h) to 21.29µm (120h). However, at P3, the nucleus length was 26.35µm (24h) and 25.22µm (120h). This information could be important for future application and use of feline BM-MSCs.(AU)

As células tronco mesenquimais são utilizadas na terapia de várias doenças na medicina humana e veterinária. As células tronco foram isoladas da medula óssea de gato, entretanto, existem poucos dados referentes a morfologia e não existem informações sobre a morfometria das células tronco isoladas da medula óssea. Os objetivos do presente estudo foram o isolamento, avaliação do crescimento, potencial de diferenciação e caracterização morfológica e morfométrica das células mesenquimais de gato isoladas de medula óssea. A diferenciação in vitro foi realizada para confirmar a multipotencialidade das células mesenquimais de gato (diferenciação em osteoblastos, condrócitos, adipócitos). As células mesenquimais foram mantidas em cultivo para avaliações morfológica e morfométrica. As células foram coradas e observadas em microscopia ótica. As mensurações foram realizadas com 24, 48, 72 e 120h de cultura (primeira e terceira passagens). O teste não paramétrico ANOVA foi utilizado e as médias foram comparadas pelo teste de Tukey. O número médio de células mononucleares obtido foi de 12,29 (±6,05x106) células/mL de medula óssea. As células mesenquimais são longas e fusiformes, e escamosas com citoplasma abundante. No estudo morfométrico, observou-se aumento no comprimento médio das células durante a primeira passagem. As medidas de comprimento das células foram: 106,97±38,16µm e 177,91±71,61µm, respectivamente, na primeira e terceira passagens (24 horas). As medidas de largura das células foram: 30,79±16,75 µm e 40,18±20,46 µm, respectivamente, na primeira e terceira passagens (24 horas). O comprimento do núcleo na primeira passagem aumentou de 16,28µm (24h) para 21,29µm (120h) e na terceira passagem foi de 26,35µm (24h) para 25,22µm (120h). As informações são importantes para futuras aplicações e uso da célula mesenquimal de gato.(AU)

Isolation and culture of human bone marrow mesenchymal stem cells without lysing the erythrocytes / 中国综合临床

Objective To observe the effects of red blood cell lysis buffer on the isolation and culture of human bone marrow mesenchymal stem cells (hMSCs) in vitro.Methods Twenty-two bone marrow samples were randomly divided into 2 groups,including 11 samples of crushed red blood cell prepared with Tris-NH4Cl red blood cell lysis agent and 11 samples prepared without red blood cell lysis agent.The intervals from primary generation to 1,2 and 3 passages and the time of expansion to l07 cells were compared between the two groups.Results The times for P1,P2 and P3 passage was ( 9.3 ± 4.9 ) days vs.( 7.2 ± 1.0 ) days,( 14.4 ±4.7) days vs.( 14.5 ± 3.5 ) days,and ( 18.5 ± 5.0 ) days vs,( 20.1 ± 4.4 ) days,respectively,in crushed red blood cell group and non-crushed red blood cell group.The differences were not significant ( t =1.39,t =0.06,t =0.80,P ＞ O.05 ).The time for expansion to 107 cells in two groups was not significantly different ( t =0.80,P ＞0.05).Conclusion Tris-NH4Cl agent in red blood cell lysis has no significant effects on hMSCs isolation,culture and cell proliferation,which indicates erythrocyte lysing may be not an independent step for hMSCs isolation and expansion in vitro.

Isolation and purification of pancreatic islets in adult donors / 中华内分泌外科杂志

Objective To investigate the methods and feasibility of islets isolation and purification,and to get more islets with high purity and good function for clinical transplantation.Methods After being weighted,human pancreatic islets were isolated with type Ⅴ collagenase,and purified by Ficoll's discontinuous density gradient centrifugation.The yield and purity of islets were evaluated by dithizone(DTZ) staining under microscope,and the viability was assessed by insulin release assay in vitro.Results The average number of islets was about 3 600 ± 447 per gram pancreas after isolation and it was about 2140 ±207 after purification with more than 70％ purity.2,4,6 days after islet cell culture,the basal insulin concentration of the culture medium was measured,and it was 3.302 ± 1.63,3.504 ±1.10,and 2.921 ±1.13 (mIU/L/100 islets) respectively.Conclusion Collagenase digest and Ficoll's discontinuous density gradient centrifugation are effective methods for isolation and purification of human pancreatic islets.

Efficient isolation of bovine keratocytes utilizing two step enzymatic digestion / 中华实验眼科杂志

Background Efficient and lowcost way to isolate keratocytes is helpful for research on cornea.Either relatively expensive or inefficient is the shortage of those means now applied,while raising the keratocytes through passage will change the phenotype of them quickly.Our aim is to approach the way getting keratocytes effectively utilizing modified two step enzymatic digestion by type I collagenase. Objective To evaluate the effect of isolating the bovine keratocytes utilizing two step enzymatic digestion and observe the morphological changes of the keratocytes during cultivation in vitro. Methods Keratocytes were isolated from bovine corneas using 0.5 mg/mL and 1 mg/mL type I collagenase digestion.The harvesting rate and viability rate of the primary keratocytes were evaluated.During the primary cultivation in vitro,the morphological changes of the keratocytes and their F-action distribution were observed.Results(1)The extracellular matrix of the bovine corneas were almost dissolved by the two step enzymatic digestion,followed the keratocytes completely isolated from the solid matrix.The amount of the harvested keratocytes from each cornea was(2.11±0.15)X106 on average while the viability rate was(91.69±3.73)% and the inoculation rate Was(81.20±1.25)%.(2)The primary keratocytes attached and spreaded out with dendritic and stellate morphology.After 3 days cultured,the branches of the keratocytes were contacting and formed networks.The F-actin detected by phalloidin binding showed a limited cortical localization. Conclusion (1)The method of two step enzymatic digestion can make the extracellular matrix of bovine cornea stroma completely degraded with the advantages in high efficiency of harvesting keratocytes and high cell viability and relatively simple manipulation. (2) The primary bovine keratocytes have dendritic morphology and with limited F-action distribute in cellular cortex.

Comparision in the yield of fetal nucleated red blood cell between the first-and second-trimester using double density gradient centrifugation / 대한산부인과학회지

OBJECTIVE: The aim of our study was to make a practical comparative evaluation of the first and second trimesters in order to determine the period during which a higher yield of fetal nucleated red blood cells (FNRBCs) can be obtained. METHODS: NRBCs were isolated from maternal blood during the first and second trimesters of pregnancy using double Percoll gradients with different osmolarities. Magnetic activated cell sorting was performed with Kleihauer-Betke stain. We isolated fetal NRBCs from 10 mL of samples of maternal blood and determined fetal sex and fetal aneuploidy by fluorescence in situ hybridization (FISH). RESULTS: The average number of NRBCs was 9.85 in samples obtained during the first trimester and 14.88 in samples obtained during the second trimester (P=0.07). The average number of NRBCs with Y chromosome signals was 5.73 in the first trimester and 8.22 in second trimester (P=0.56). However, the percentage of NRBCs with Y chromosome signals in the first trimester (70.6%) was significantly higher than in the second trimester (59.8%) (P=0.049). We diagnosed the blood samples from 7 pregnant women having fetal aneuploidy using this method and the number of NRBCs was 18.4. CONCLUSION: The method using Percoll osmolarity and a double density gradient system may be a very useful method for separation of NRBCs in the first trimester of pregnancy and also in the second trimester.

Research progress in hepatic stellate cells / 解剖学报

Hepatic stellate cells (HSCs) are a group of nonparenchymal cells of liver, and play an important role in lipid, especially vitamin A-storing, extracellular matrix synthesis, microcirculation regulation of hepatic sinusoid, etc. They are also closely related to lots of liver diseases, e. g. hepatofibrosis. In this review, the latest research advance on HSCs including their isolation, identification, proliferation and regulation, and role in liver regeneration, hepatofibrosis, and so on is summarized.

Biological Characteristics of Human Colon Cancer Cell Line CW-2 Stem Cells / 中国医科大学学报

Objective To isolate cancer stem cells from human colon cancer cell line CW-2,and observe the biological characteristics.Methods The percentage of CD44＋EpCAM＋ cancer stem cells which were isolated by multi-combination method was evaluated by flow-cytometry.The biological characteristics of the stem cells were analyzed by cell cycle analysis,in vitro invasion assay and in vivo tumorigenicity assay.Results The percentage of CD44＋EpCAM＋ cancer stem cells was 89.57%,and their capabilities of generation,invasion,tumorigenicity were higher than non-cancer stem cells（P 0.05）.Conclusion CD44＋EpCAM＋ cancer stem cells showed low generation,high invasiveness,high tumorigenesis,and they could be isolated by multi-combination.

Application of a modified method for stem cell isolation from lipoaspirates in a basic lab.

Aim Lipoaspirate, a wasted by product from liposuction procedure recently has been shown to contain abundant mesenchymal stem cells (MSCs). MSCs have been studied in many research areas to regenerate many cell lineages including, myogenic, cardiomyogenic, and angiogenic lineages. The large quantity of MSCs in lipoaspirate, makes it an attractive source for stem cells used in research and clinical applications. A simplified method which is suitable to be performed in a basic laboratory will facilitate development of stem cell research in developing countries. Therefore the outcomes from this study are expected to encourage the progress of stem cell research in Indonesia. Methods Lipoaspirate was digested using collagenase type I, followed by a basic filtration method. Purification of MSCs was done by cell culture for 2-3 days followed by supernatant removal. To confirm the homogenous population of MSCs, an analysis using flowcytometry was performed based on the MSCs minimal criteria developed by Mesenchymal and Tissue Stem Cell Committee of the International Society of Cell Therapy. Resuts MSCs were able to be obtained at 16.41 ± 8.22 x 108 cells per 120 ml lipoaspirate. The cultured cells showed fibroblastic morphology which is characteristic for MSCs and were able to be purified from non-MSCs cells. This was confirmed by flowcytometry assay showing expression of CD105 and the absence of HLA-Class II, CD 45, CD 34, CD14, and CD19. Conclusions This study has shown that it was feasible to isolate messenchymal stem cell from human lipoaspirate. The procedure was practicable to be performed within a basic laboratory.

Isolation of hepatocytes:a review of current methods / 国际外科学杂志

Due to the increasing use of hepatoeytes,the demand of hepatoeytes is increasing consistently in basic clinical research.The application of rational and efficient method of hepatoeyte isolation is a prereq-uisite for its application.At present,there are many methods of hepatoeyte isolation.This review summarizes the latest progress on them.

Isolation, culture and identification of hmnan epithelial stem cells / 重庆医科大学学报

Objective:To explore a method for isolation and culture of human epidermal stem cells. Methods:The Epidermal stem cell were isolated by adhering to collagen typeⅣafter obtained by digesting human foreskin with DispaseⅡand Tryp and culture in vitro in K-SFM. The expressions of?1-integrin and keratin 19 (K19) in epidermal stem cell were detected with immunocytochemical methods,and the colony forming efficiency was also studied. Keratinocytes were served also detected. Results:It was revealed by histologieal observation that colonieswere formed 24 hours after inoculation. The isolated and cultured cell cloning efficiency was higher than that of the control group. Positive expression of?1-integrin and K19 of cultured cells was detected by immunocytochemistry. Conclusion:Adult epidermal stem cells could be successfully isolated and cultured by adhension with typeⅣcollagen and culture with K-SFM

Isolation, culture and identification of human epidermal stem cells / 上海第二医科大学学报

Objective To explore a method for isolation and culture of human epidermal stem cells. Methods Epidermis was obtained by digesting human foreskin with Dispase Ⅱ and Trypsin-EDTA.After suspension on the epidermal stem cell medium (ESCM), these single epidermis cells were inoculated onto human collagen Ⅳ-coated flasks and cultured at 37 ℃ in a humidified atmosphere containing 5% CO_2 for 10 min. The nonadherent cells were rinsed off 10 min after inoculation, and the adherent cells continued to be cultured after enriching and abstraction by type Ⅳ collagen. The cell growth was observed through inverted microscope, and the cell cloning efficiency and time of clone sustain were also detected. Immunocytochemistry was used to observe the expression of ?_1-integrin and keratin 19(K19). Keratinocytes were served as controls. Results It was revealed by histological observation that colonies were formed 24 hours after inoculation. The isolated and cultured cell cloning efficiency was higher and the time of clone sustain was longer than that of the control group. Positive expression of ?_1-integrin and K19 of cultured cells was detected by immunocytochemistry. Conclusion Adult epidermal stem cells could be successfully isolated and cultured by adhension with type Ⅳ collagen and culture with ESCM.

Isolation of transfected fibroblast clones for use in nuclear transfer and transgene detection in cattle embryos

Transgenesis in cattle has provided numerous opportunities for livestock production. The development of nuclear transfer (NT) technology has improved the production of transgenic livestock. However, the isolation of pure colonies from a single transfection event remains laborious and can be a constraint in the production of transgenic livestock. We used 96-well cell culture plates to isolate cell lineages obtained from a single fibroblast transfected with the pCi-Neo plasmid. Since single mammalian cells do not grow well in fresh medium, we evaluated the use of conditioned medium. The neomycin phosphotransferase gene was detected in isolated colonies and NT embryos were produced from these cells. Multiplex-PCR assays were performed to detect the transfected fragment as well as autosomal satellite DNA in single NT embryos. This approach provided a reliable method for isolating transfected mammalian cells and for diagnosing the incorporation of desirable vectors in NT embryos. This method can reduce the time and cost of transgenic livestock production.

Isolation and Purification of Kupffer Cells from Rat Liver / 中国普外基础与临床杂志

Objective To explore a simple, effective and stable method for the isolation and purification of Kupffer cells from rat liver, enabling further study on the structure and function of these cells in vitro. Methods After laparotomy, a catheter was inserted into the portal vein and secured with artery clamp. Then, the rat liver was perfused and digested with solution Ⅰ and solution Ⅱ containing 0.05% collagenase Ⅳ respectively. The cell suspension was centrifuged with isopycnic sedimentation in a two-step Percoll gradient to harvest Kupffer cells. The isolated Kupffer cells were purified by selective adherence after 30 min of cultivation, and identified by evaluation of phagocytosis of India ink and peroxidase staining with DAB through light and electron microscopy. Results It was verified that the viability of isolated Kupfffer cells was more than 90% through Trypan blue staining. Those Kupffer cells could attach to plastic quickly and phagocytose ink, and had the appearance of "fried eggs" in positive peroxidase staining with a purity of 95%. Under the light microscopy, the appearance of newly isolated Kupffer cells was round with uniform shape and size. After two days of culture, Kupffer cells appeared to distend with irregular or stellate shape. The typical features were observed in the transmission electron micrographs. There were numerous pseudopods and occasional cup-like indentations in the cell membrane of Kupffer cells. The cytoplasm contained numerous types of lysosomes and other phagocytotic vesicles. Conclusion The method for isolating and culturing Kupffer cells in this study is effective and stable, and the biological characters are preserved in the cultured cells.