فهرست مطالب arezoo rezaie nezhad zamani

 The need for on-demand biological products has been raised during the last decades. To prepare ready-to-use organ-related products, it necessitates a bulk cell reservoir. In this regard, stem cells, especially mesenchymal stem cells, have numerous therapeutic properties in tissue repair. Therefore, the advent of novel cell banking systems is inevitable to maintain cells ready-to-use.

 In the current study, the cryoprotective effects of alginate-gelatin were investigated on human mesenchymal stem cells after seven days.

 Mesenchymal stem cells were classified into two groups; the Control and Encapsulated cells. Cells were encapsulated by using the mixture of alginate (1% v/v) and 2% gelatin and 1% CaCl2, a cross-linker, and vitrified in liquid nitrogen in freezing medium containing 10% dimethyl sulfoxide. After seven days, mesenchymal stem cells were thawed and decapsulated by 0.01 M sodium citrate solution. The cell survival rate was monitored by using MTT assay. Flow cytometry analysis of annexin-V/PI was used to determine the number of apoptotic cells.

 These data showed that encapsulation had a superior effect on maintaining cell viability after the freeze-thaw procedure compared to the control group (P < 0.05). We found a significant decrease in the number of early- and late-stage apoptotic cells in mesenchymal stem cells inside the alginate-gelatin microspheres seven days after deep freezing (P < 0.05). As expected, cell cycle analysis specified the lack of dynamic activity in cells from both groups. Cells at phases S and G2/M reached zero.

 These findings showed that encapsulation of human mesenchymal stem cells with alginate-gelatin microspheres could reduce deep freeze/thaw insult and decrease the detrimental effect of cryoprotectant compared to non-capsulated cells.

Today, there is an urgent need to develop a three-dimentional culture systems mimicking native in vivo condition in order to screen potency of drugs and possibly any genetic alterations in tumor cells. Due to the existence of limitations in animal models, the development of three dimensional systems is highly recommended. To this end, we encapsulated human colon adenocarcinoma cell line HT29 with alginate-poly-L-lysine (Alg-PLL) microspheres and the rate of epithelial-mesenchymal transition was monitored.

Cells were randomly divided into three groups; control, alginate and Alg-PLL. To encapsulate cells, we mixed HT-29 cells (1 × 106) with 1 mL of 0.05% PLL and 1% Alg mixture and electrosprayed into CaCl2 solution by using a high-voltage power. Cells from all groups were maintained at 37˚C in a humidified atmosphere containing 5% CO2 for 7 days. Cell viability was assessed by MTT assay. To monitor the stemness feature, we measured the transcription of genes such as Snail, Zeb, and Vimentin by using real-time PCR analysis.

Addition of PLL to Alg in a hallowed state increased the cell survival rate compared to the control and Alg groups (P<0.05). Cells inside Alg-PLL tended to form microcellular aggregates while in Alg microspheres an even distribution of HT-29 cells was found. Real-time PCR analysis showed the up-regulation of Snail, Zeb, and Vimentin in Alg-PLL microspheres compared to the other groups, showing the acquisition of stemness feature (P<0.05).

This study showed that hallow Alg-PLL microspheres increased the epithelialmesenchymal transition rate after 7 days in in vitro condition. Such approaches could be touted as appropriate in vitro models for drug screening.