Adipose Stem Cells with Conditioned Media for Treatment of Acne Vulgaris Scar

This study was to investigate the effect of subcutaneous injection of the adipose stem cells (ASCs) with conditioned media (CM) in the treatment of acne vulgaris scar. We used Adult male New Zealand white rabbit ears as an animal model and induced acne formation by Kignman method. Adipose tissue was isolated and harvested from the scapula of rabbits, and ASCs were cultured and expanded until passage 1. There have four groups in our experiment, include phosphate buffered saline (PBS), ASCs with PBS (ASC + PBS), CM, and ASCs with CM (ASC + CM) group. This solution of 0.6 ml injected to subcutaneous in each group. ASC + PBS and ASC + CM groups were containing ASCs of 5.0 × 106 cells/ml. We analyzed the treatment of 4 groups to scar tissue after 2 and 4 weeks by hematoxylin and eosin stain, immunohistochemistry, and RNA expression level of tumor necrosis factor-α (TNF-α), interleukin-1α (IL-1α), and matrix metalloproteinase-2 (MMP-2). Also, the expression of keratin 16 (K16) was detected by western blot analysis. H&E stain showed that infiltration of inflammation cells was significantly reduced at 2 and 4 weeks, as well as re-epithelialization was improved in the ASC + CM group. The ASC + CM gourp was reduced both expression levels of TNF-α, IL-1α, and MMP-2 and K16 protein level. In conclusion, the ASCs with CM has a significant curative effect on acne vulgaris scar, more to the point, the CM has a key role on treatment. It could be applied to a therapeutic approach to regenerate to treat acne vulgaris scar.

Lactoferrin Constitutively Enhances Differentiation of Osteoblastic MC3T3-E1 Cells in Vitro

During bone remodeling, there is requirement of differentiation of osteoblastic cells. Previously, we identified proteins differentially expressed in soft tissue during bone healing. Of these proteins, we focused the effect of LTF on differentiation of osteoblast. In order to analyze the osteogenic ability of LTF, we treated conditioned media collected from human LTF-stably transfected HEK293T cells into osteoblastic MC3T3-E1. The results showed that the activity and expression of alkaline phosphatase were increased in MC3T3-E1 cells treated with conditioned media containing LTF in dose- and time-dependent manner. At the same time, we observed the significant increase of the expression of osteoblastic genes, such as ALP, BSP, COL1A1, and OCN, and along with matrix mineralization genes, such as DMP1 and DMP2, in LTF conditioned media-treated groups. Moreover, the result of treating recombinant human LTF directly into osteoblastic MC3T3-E1 showed the same pattern of treating conditioned media containing LTF. Our study demonstrated that LTF constitutively enhances osteoblastic differentiation via induction of osteoblastic genes and activation of matrix mineralization in MC3T3-E1 cells.

Effects of conditioned media from human amniotic epithelial cells on corneal alkali injuries in rabbits

This study was performed to evaluate the effects of conditioned media (CM) from human amniotic epithelial cells (HAECs) on the corneal wound healing process. Eighteen rabbits (36 eyes) were used and randomly assigned to three groups according treatment: CM from HAECs (group 1), vehicle alone (group 2), and saline (group 3). Corneal alkali injuries were induced with 1 N sodium hydroxide. Each reagent used for treatment evaluation was injected into the dorsal bulbar subconjunctiva and the area of the corneal epithelial defect was measured every other day. Two animals from each group were euthanized at a time on days 3, 7, and 15, and the cornea was removed for histological examination. The sum of the epithelial defect areas measured on day 0 to day 6 as well as day 0 to day 14 in group 1 was significantly smaller than those of other groups. Histological examination revealed that the group 1 corneas had less inflammatory cell infiltration and showed more intact epithelial features compared to the other groups. These results suggest that CM from HAECs promote corneal wound healing in rabbits.

Effect of Converted Regulatory T Cells with Renca Conditioned Media on Suppressive Profile of Allo-immunity in Mice / 中山大学学报(医学科学版)

[Objective]This study was designed to investigate capability of the conditioned media that originated from Renca cells to convert CIM~+ CD25~- T cells into CD4~+ CD25~+ T cells,which can exert immunosuppressive effect on effector T cells in vitro and in vivo.[Methods]The common media were mixed with the conditioned media at different ratios,and fresh enriched CD4~+ CD25~- T cells with MACS were cultured in mixed media for 7 days.At end-point of culture,the cells were collected and detected phenotypes in flow cytometer.Moreover,we detected immunosuppressive effect of converted CD4~+ CD25~+ T cells on effeetor T cells proliferation in one-way mixed lymphocytes reaction by using CCK-8,and we observed survival time and histology of grafts.The delayed type hypersensitivity was determined 14 days after transplantation.[Results]The mixed media could increase ratio of CD4~+ CD25~+ Foxp3~+ T cells in conditioned media ratio-dependent(P＜0.05),compared with control groups,when the mixed media contained no mote than 75% of conditioned media.The converted CD4~+ CD25~+ T cells significantly suppress proliferation of effector T cells in vitro,and prolong survival time of grafts,which were(29.6±1.4)d in converted CD4~+ CD25~+ T cells treated groups(P＜0.05),compared with that in untreated groups(9.8±0.6 d)or PBS treated groups(10.9±0.6 d).Moreover,delayed-type hypersensitivity reaction were conducted at day 14 after transplantation in the recipients,and the results showed that less pad swelling in the group treated with converted CD4~+ CD25~+ T cells than other control groups was found,according to measurement of pad swelling.In addition,progressed to complete necrosis of grafts were exhibited in the mice treated with PBS and untreated mice,whereas better healing of grafts and less lymphocytes infiltration were displayed in the mice treated with converted CD4~+ CD25~+ T cells,which were similar to the mice treated with natural regulatory T cells.[Conclusion]The converted CD4~+ CD25~+ T cells with Renca conditioned media play suppressive role in vitro and in vivo.

Studies on development of serum-free conditioned media using Vero cells and DMEM with controlled concentration of glucose and pyruvate / 대한산부인과학회지

OBJECTIVE: The purpose of this study was to examine in vitro development of early preimplantation mouse embryos in various kind of serum-free conditioned media (SF-VCM) manufactured from DMEM cultured with Vero Cells. METHODS: A total of 846 two-cell mouse embryos were cultured in different kind of SF-VCM. SF-VCMs were divided into SF-VCM-10, -30 and -50 by media volume using DMEM #1 media, and divided into SF-VCM #1, #2 and #3 by controlled concentration of glucose and pyruvate (manufactured by DMEM #1: mixed three volume of DMEM-G (DMEM with glutamine without glucose and pyruvate) and one volume of DMEM-GGP (DMEM with glutamine, glucose, pyruvate), #2: mixed same volume of DMEM-G and DMEM-GGP and #3: mixed one volume of DMEM-G and three volume of DMEM-GGP, respectively). Experimental groups were mainly added 10% SSS, and 20% hFF was added to only Control group co-cultured with Vero cells. Development of embryos was observed every 24 hours. Results between different groups were analyzed using Chi-square test, and considered statistically significant when P-value was less than 0.05. RESULTS: In vitro developmental rate by each cleavage stages of mouse embryos cultured in SF-VCMs with a various volumes were significantly (P<0.05) higher in SF-VCM-30 (morula< or =: 97.2%, Blastocyst (BL)< or =: 97.2%, Hatching BL< or =: 82.2%) than other groups. In the rate of development on in vitro co-culture vs. a various SF-VCMs manufactured by DMEM controlled concentration of glucose and pyruvate, Group I (SF-VCM #1) was higher than other groups in each cleavage stages (morula< or =: 98.1%, Blastocyst (BL)< or =: 97.1%, hatching BL< or =: 81.7%, respectively). Moreover, specially, in the developmental rate into the hatching blastocyst < or = after 96 hours in vitro culture, Group I (81.7%) was significantly higher than control group (67.6%, P<0.05). CONCLUSION: SF-VCM #1 manufactured by volume of 30 mL DMEM #1 media cultured in vitro for 48 hours in 250 mL flask was the most effective on in vitro developmental rate of mouse preimplantation embryos. Therefore, it is expected that SF-VCM #1 has application to human IVF-ET.

Regulation of cementoblast differentiation and mineralization using conditioned media of odontoblast / 대한치주과학회지

For the regeneration of periodontal tissues, the microenvironment for new attachment of connective tissue fibers should be provided. At this point of view, cementum formation in root surface plays a key role for this new attachment. This study was performed to figure out which factor promotes differentiation of cementoblast. Considering anatomical structure of tooth, we selected the cells which may affect the differentiation of cementoblast - Ameloblast, OD11&MDPC23 for odontoblasts, NIH3T3 for fibroblsts and MG63 for osteoblasts. And OCCM30 was selected for cementoblast cell line. Then, the cell lines were cultured respectively and transferred the conditioned media to OCCM30. To evaluate the result, Alizarin red S stain was proceeded for evaluation of mineralization. The subjected mRNA genes are bone sialoprotein(BSP), alkaline phosphate(ALP), osteocalcin(OC), type I collagen(Col I), osteonectin(SPARC ; secreted protein acidic and rich in cysteine). Expression of the gene were analysed by RT-PCR. The results were as follows: 1. For alizarin red S staining, control OCCM30 didn't show any mineralized red nodules until 14 days. But red nodules started to appear from about 4 days in MDPC-OCCM30 & OD11-OCCM30. 2. For results of RT-PCR, BSP mRNAs of control-OCCM30 and others were expressed from 14 days, but in MDPC23-OCCM30 & OD11-OCCM30 from 4 days. Like this, the gene expression of MDPC23-OCCM30 & OD11-OCCM30 were detected much earlier than others. 3. For confirmation of odontoblast effect on cementoblast, conditioned media of osteoblasts(MG63) which is mineralized by producing matrix vesicles didn't affect on the mineralized nodule formation of cementoblasts(OCCM30). This suggest the possibility that cementoblast mineralization is regulated by specific factor in dentin matrix protein rather than matrix vesicles. Therefore, we proved that the dentin/odontoblast promotes differentiation/mineralization of cementoblasts. This new approach might hole promise as diverse possibilities for the regeneration of tissues after periodontal disease.

Effects of buthionine sulfoxine on the redox state of human umbilical vein endothelial cells cultured with K562 cells conditioned media / 中国药理学通报

Aim To investigate the role of K562 cells conditioned media on redox state of human umbilical vein endothelial cells (HUVECs) and the role of buthionine sulfoxine(BSO), a selective inhibitor of ?-glutamylcysteine synthetase, on HUVECs cultured with K562 cells conditioned media. Methods Glutathione(GSH)、oxidized glutathione (GSSG)、NADP~+、NADPH concentration and the viability of HUVECs under various conditions were determinated. Results GSSG、GSH、NADP~+、NADPH concentration of HUVECs increased when HUVECs were cultured with K562 cells conditioned media. The inhibition of HUVECs growth by Bso enhanced when K562 cells conditioned media were used at the same time. Conclusion Under the effection of chronic myelogenous leukemia cells conditioned media, endothelial cells may be more sensitive to BSO.