Assessment of hepatocellular function within PEG hydrogels

http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6TWB-4KXDWJ4-3&_coverDate=01%2F31%2F2007&_alid=508921244&_rdoc=1&_fmt=&_orig=search&_qd=1&_cdi=5558&_sort=d&view=c&_acct=C000059607&_version=1&_urlVersion=0&_userid=4420&md5=0a6bb7dc1ed695e1446012bbc052fe98


Tissue engineered liver has the potential to replace organ transplantation; however, due to the complexity of liver structure and function, fabricating the hepatic tissue is very challenging. This paper illustrate the utility of photopolymerizable poly (ethylene glycol) hydrogels for 3D encapsulation of heptic cells.
In this paper, they studied the survival and function of hepatic cells within the PEG hydrogel platform. They seeded bipotential mouse embryonic liver (BMEL) and Fibroblasts making Hepatocytes and Fibroblasts co-cultures (were seeded at a density of 5.0X105 cells/well, absorbed with 0.13mg/mL Collagen-1 extract from rat tail tendons) in PEG hydrogel platform, then, performed encapsulation of BMEL cells and primary hepatocytes. The cell viability was examined using calcein AM and ethidium homodimer fluorescent stains; also, performed RT-PCR to analysis gene expression and short interfering RNA transfection. To assess hepatocellular function and albumin release, used ELISA method, an enzyme linked immunosorbent assay. Found that the encapsulated BMEL aggregates and displayed a substantial induction of both albumin and alcohol dehydrogenase, which are both important markers of hepatocyte differentiation. So, this exhibited that BMEL cells can differentiate within PEG hydrogel plateform. Survival of PEG hydrogel encapsulated BMEL cells, for monodispersed cell culture, after one hour the viability was high of a 95%. However, after 3 days of post-encapsulation, the viability dropped down to 25%. As for pre-aggregated BMEL cells showed a significant increase in viability. This demonstrated the cell-cell interactions in maintenance of BMEL viability.
Also, to demonstrate the efficiency of siRNA-mediated gene knockdown for the hydrogel encapsulated BMEL cells, they examined slicing efficiency for a representative gene, lamin A. Found that the hydrogel encapsulation does not directly affect the maintenance of gene knockdown. This shows the compatibility of BMEL cell differentiation with hydrogel culture and the capacity to regulate gene expression through RNA interference.

I choose this paper because my final project is testing the hepatocyte’s ability to survive in agarose gel culture, which is similar with what this paper described. We tested the expression of albumin by RT-PCR, which is similar with what the paper did. Also, we measured the cell viability and the albumin production rate each time frame per cell. In our project, we used partially collagen I mixture with agarose to make the 3D culture. We observed that the culture with collagen I tend to have cells clumping together, and have better survival rate than the agarose only culture. This is also observed in this paper where they demonstrated the cell-cell interactions in maintenance of BMEL viability.

Injectable Liver: A Novel Approach Using FibrinGel as a Matrix for Culture and IntrahepaticTransplantation of Hepatocytes
TISSUE ENGINEERINGVolume 11, Number 11/12, 2005
http://www.liebertonline.com/doi/pdf/10.1089/ten.2005.11.1718

In this paper, the researchers tried to focus on cell transplantation and tissue engineering techniques with liver cells as experimental therapies for some liver diseases. They made fibrin-based gel matrix as carrier for hepatocytes in culture. Then they developed a direct injection technique to take hepatocytes harvested from rats. After cell isolation, they did PKH26 labeling. Adult Sprague-Dawley rats were used as organ donor and recipients. For cell transplantation, a minilaparotomy was performed; the cell-matrix mixture was injected directly to the organ. After different time harvest, animal were killed and liver were removed and to take histological investigation. DNA quantification was preformed and cell numbers showed the viability. In another hand, they did RNA extraction, cDNA synthesis and PCR (RT-PCR). The explanted liver tissue was snap frozen in liquid nitrogen. Cytospins, fixtion, histology, and immunohistochemistry were preformed on the tissue. In the end, statistical analysis provide the results.

From the experiments they did above, they drew some results. Cell numbers were assessed by DNA content, they showed the viability of the cells is good, which means fibrin matrix is an appropriate environment for hepatocytes. Fluorescence microscopy of the liver was performed to identified PKH26 labeled cells. RT-PCR and IH showed preservation of hepatocytes and hepatic stellate cells into the host liver. Direct injection technique of the fibrin gel-immobilized hepatocytes is technically feasible. They concluded that fibrin gel immobilization is an good tool for develop tissue engineering artificial liver system. They also discussed the fibrin glue as a matrix supportive of hepatocytic differentiation, using fibrin glue as a carrier for injectable liver, and remolding of liver tissue after injection of fibrin-hepatocyte mixture.

I chose this paper, since it’s really similar to what we are doing in the final project. These German researchers used fibrin gel as matrix, we did use agarose gel to culture cells. They did future steps to seed the culture into rats and gave a normal better modeling environment, which is more worth us to think about. We are performing most of their cell analysis techniques, which we can watch as references such as RT-PCR, Cell counting and immunohistochemistry. We can do a lot of comparison and contracts between our projects and theirs. Their results are good, which mean other gel than collagen can work with liver cells. This gives us motivation to see our result on agarose gel. In the end, their discussions are also very advisable.

Development of a Closed Bioreactor System for Culture of Tissue-Engineered Skin at an Air–Liquid Interface

(This is Terry posting on the behalf of Elena, who's having blogger problems.)

Before autologous tissue-engineered skin providing a reasonable barrier can progress to the clinic, it is needed to design bioreactor that can construct cells at an air-liquid interface. This article investigates and compares the designs for continuous as well as batch-reaction bioreactors. A comparison between continuous perfusion with medium versus changing medium every few days was also used to determine which method would better support metabolic activity.

Four different scaffolds were used in this study: acellular deepidermized human dermis (DED), electrospun polystyrene (PS; 0.8–1.0 mm thick), a composite of electrospun poly-DL-lactide fiber and polystyrene (PDLA/PS) (0.8–1.0 mm thick), and a commercially available nonwoven scaffold (Azowipes). Normal human keratinocytes and fibroblasts were isolated and cultured and the viable cell density was assessed by MTT-ESTA (an assay based on the conversion of MTT to a colored formazan end product by intracellular dehydrogenase activity). Viability measurements by MTT indicated that fibroblast and endothelial single cultures preferred submerged culture conditions, whereas an air–liquid interface gave greater total cell viability for the single culture of keratinocytes and fibroblast–keratinocyte cocultures compared with submerged cultures. MTT measurements showed that total cell viability of fibroblast–keratinocyte cocultures was significantly higher in PS and PDLA/PS compared with DED.

Results also indicated that the cell viability of tissues cultured under continuous perfusion was significantly higher than that of tissues cultured under batch-fed culture (Fig. 5). It was noticeable that total cell viability was almost 2-fold higher for PS electrospun scaffolds with continuous feed versus DED with continuous feed. In the case of keratinocytes this may result in cells failing to proliferate or it may induce premature abnormal differentiation. Thus, continuously perfused medium is likely to be more effective than frequent changes of static culture medium every few days.

Overall, the bioreactor developed in this investigation shows several technical and operational advantages that will be of significance for skin tissue-engineering research. These advantages include: a completely closed system, a bioreactor that can be operated easily under sterile conditions, a design the potential for parallel connection of individual chambers, allowing for multiple experimental reactors to be stacked vertically and therefore minimizing culture space, and a bioreactor with multiple chambers to allow for simultaneous experiments.

I chose this article due to its relevance to cell-culture techniques used during our lab. The feeding technique use din lab consists of changing the medium every few days, however in this report, it is suggested that a continuous perfusion of medium resulted in an increased cell viability. In addition, this investigation focuses on an air-liquid interface cell culture, which would be needed in tissue-engineered skin to be produced for clinical use. This aspect relates to our project of tissue engineering dermis and can provide insight as to possible future work that could provide better results.