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Containing cells in vitro tissue-engineered corneal stroma method_2606

By Carl Wood,2014-10-30 14:11
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Containing cells in vitro tissue-engineered corneal stroma method_2606

Containing cells in vitro tissue-engineered corneal stroma

    method

     Abstract Objective: To study the corneal stroma in vitro tissue engineering approach. Methods: The co-enzyme detergent

    method of dealing with acellular porcine corneal stroma, in

    the acellular porcine corneal stroma in vitro inoculation materials rabbit corneal stromal cells, and rabbit corneal stromal cells to PKH26 fluorescent marker, in vitro tissue engineering corneal stroma. Inverted fluorescence microscope;

    HE staining optical microscope; scanning electron microscope. Results: The use of xenogenic acellular corneal stroma Organization for scaffold materials in tissue engineering built to approximate a normal corneal stroma with a three-

    dimensional structure of corneal stroma. Conclusion: The

    acellular porcine corneal stroma material for stents, the use of tissue engineering techniques can be successfully built to contain in vitro tissue-engineered corneal stroma cells in the organization.

     Key words Tissue engineering of corneal stroma to build

     In vitro construction of cellular tissue engineering corneal stroma

     Abstract AIM: To study the in vitro construction process of cellular tissue engineering cornea stroma. METHODS: Porcine cornea stromata were processed by serial digestion of trypsin, Dnase and Rnase. Then rabbit corneal stroma cells were raised in it, marked by fluorchrome pkh26. Tissue engineering cornea stromata were constructed in vitro. Inverted fluorescence microscope, HE staining light microscope

    and scanning electron microscope were used in the observation.RESULTS: In vitro constructed tissue engineering

    cornea stroma had the similar three diamension construction with the normal corneal stroma.Conclusion: Acellular porcine cornea stroma materials can be used as carrier material to construct tissue engineering cornea in vitro.

     * KEYWORDS: tissue engineering; corneal stroma; construction

     0 Introduction

     The lack of donor cornea limits the wide range of corneal

    transplants carried out, building engineering cornea donor cornea to replace the materials for clinical transplantation to provide more extensive material is the corneal tissue engineering research hot spots. How to construct tissue-

    engineered corneal stroma [1] is an important part. Stroma constitutes the main part of the cornea, accounting for 90% of corneal thickness, mainly by collagen fibers, corneal stromal cells and extracellular matrix composition. In lamellar keratoplasty in corneal stroma of the donor pathology played a

    major role in prognosis [2], and therefore in vitro-engineered

    corneal stroma has important clinical value.

     1 Materials and methods

     1.1 Materials of York to take fresh porcine full-

    thickness cornea. With Tris-EDTA (10m mmol / L Tris, 5m mmol /

    L EDTA) overnight, after, 10g / L detergent Triton 40C and then overnight. 2.5g / L trypsin digestion 37 ?, after 4h,

    and then 1:1 000 DNA enzyme-RNA enzyme digestion 4h acellular 37 ? treatment, 10g / L Triton overnight. 50mmol / L Tris-

    EDTA, triple-distilled water, washing three times shocks will be full-thickness corneal pieces cut into 0.2mm thick lamellar films. -20 ? freeze-dried. Co60 irradiation sterilization -

    20 ? to save. 1mo age of New Zealand to take a fresh young rabbits cornea, using a PBS solution containing two anti-

    carefully washed, 5min × 3 times. The whole layer of the

    cornea and then cut to remove the iris tissue, cornea epithelium, inner cortex, secondary antibody in PBS containing liquid again, rinse, 5min × 3 times. Cut the corneal tissue

    organization 1mm3 fragments by adding collagenase 625ku / L digest 30 ~ 60min after the termination of digestion, centrifugation, discard supernatant, add DMEM culture medium

    and inoculated in culture bottles, placed 37 ? incubation box

    within a culture, every 3d for liquid 1. When cell confluence, the original cultured corneal stromal cells were digested with trypsin wind and percussion into a cell suspension grown in cover slips, add 1mL placed in DMEM culture medium and incubated for hatching Box 24 ~ 48h, the cell is covered with glass tablet acetone fixed 15min, will be fixed with a good cell seeded 0.01mol / L PBS buffer, washed three times, each time 5min; 2.5mL / L Triton X-100 treatment 10 min; PBS washed

    3 times shock, each time 5min; 30ml / L H2O2 closed endogenous peroxidase 10min; PBS washed 3 times shock; an anti-drip, Ck

    (1: 150), Vim (1: 50), also set up blank control, PBS replaced by a resistance; 37 ?, 150min; of the following

    immunohistochemical staining kit according to the requirements. Dropping DAB color at room temperature 5 ~ 10min, PBS wash to terminate the color reaction. Progressively dehydrated, xylene and transparent, neutral gum Fengpian. Will be the 3rd generation of rabbit corneal stromal cells were trypsinized into single cell suspension, take 2 × 107 cells

    in the cone-shaped centrifuge tube, according to PKH26 marked with operating instructions, simple steps are as follows: The cells, after washing with PBS, centrifuged to form loose cell clumps to 1 mL diluent reagent box re-suspended; to 1.5μL

    PKH26 dye diluted by adding cells, rapid and uniform mixing, 25 ? incubation 2 ~ 5min (centrifuge tube upside down from time to time to ensure full mixing); adding equal serum

    termination reaction, incubated 1min; serum-containing

    complete medium centrifugal cleaning cells, at least three times; the cells at the desired concentration of weight hanging, inoculation.

     1.2 Methods PACM in the ultra-clean the desks of about

    12mm in diameter wafer cut into 24-well plates, the culture

    medium with DMEM soak 1 ~ 2d, abandoned to the culture medium, spare. Will be marked with PKH26 rabbit corneal stromal cells were inoculated at 4 × 106/cm2 density of the PACM on standby

    to join DMEM culture medium, placed 37 ?, 50mL / L CO2

    incubator box culture, 1 ~ 2d for liquid 1. 5d culture drawn for HE staining optical microscope observation; scanning electron microscopy; frozen sections observed by fluorescence microscopy. Reposted elsewhere in the paper for free download http://

     2 Results

     Digestion of rabbit corneal stromal cells 1 ~ 2h adherent, spindle-shaped cells, stretching the cytoplasm clear, and the nuclear center. 3 ~ 5d cells to form dense single layer, arranged very neatly, was swirling. Immunohistochemical results: positive for vimentin staining (Figure 1), negative keratin staining to prove that cellular contamination of non-epithelial cells, a single corneal

    stromal cells. Histological observation: rabbit corneal

    stromal cells showed spindle-shaped extension, in the

    acellular porcine corneal stroma in close quote attached to surface (Figure 2A). Scanning electron microscopy: rabbit corneal stromal cells were polygonal stretching acellular porcine corneal stroma in the material surface, adhesion of cells and materials, the close (Figure 2B). Can be seen acellular porcine corneal stroma material attached to red fluorescence of rabbit corneal stromal cells (Figure 2C)

     Figure 1 cultured rabbit corneal stromal cells stained

    positive for vimentin

     3 Discussion

     Construction of tissue-engineered corneal stroma the key to success is to apply an ideal carrier to grow as the extracellular matrix of corneal stromal cells to form a body

    similar to the normal structure. This vector should support cell growth, promote cell fusion, with good

    histocompatibility. Porcine and human corneal keratocytes similar structure, there is a complete extracellular matrix, basement membrane, Bowman layer and the substrate, there are a

    variety of growth factors, can induce regulate cell growth, reproduction, differentiation and proliferation. Studies have found that the immunogenicity of corneal stroma is very low, in the cell-mediated immunity, the corneal stroma accounts for

    only 1.62% [3]. Li et al [4] found that corneal and limbal epithelium, corneal fibroblasts existed between cytokine regulatory networks. Friend et al [5], Wu Jing et al [6], respectively, on porcine corneal stroma cultivated corneal epithelial cells in vitro to form a near-physiological

    conditions of the corneal epithelial tissue. Amano et al [7] In the porcine corneal matrix cultured human corneal endothelial cells and the transplant was successful. We found that acellular porcine corneal stroma material has a three-

    dimensional mesh-like structure, and good biocompatibility

    [8], but also a better therapeutic effect. Using it as stand inoculation, cultured rabbit corneal stromal cells built to contain cells in tissue engineering cornea by scanning

    electron microscopy found to contain cells, tissue engineering cornea with a three-dimensional mesh-like structure, similar

    to normal corneal architecture, may be provided to the cornea stromal cells with adequate nutrients and material exchange

    space, conducive to cell growth, proliferation and differentiation. Corneal stromal cells in acellular porcine corneal stroma material attached growing well. Observed under fluorescence microscope acellular porcine corneal stroma red fluorescent material attached to the rabbit corneal stromal cells, and further illustrates containing stromal cells, tissue-engineered corneal stromal cells contained some activity. Therefore, this experiment was successfully constructed in vitro tissue engineering of living cells

    containing the corneal stroma.

     References

     1 Vacanti CA, Vacanti JP. Bone and Cartilagere construction with tissue engineering approaches. Otolaryngol Clin North Am, 1994; 27 (1) :263-276

     2 CHEN Dong-Hong, YUAN Nan-jung. Experimental full-thickness

    lamellar keratoplasty for immunohistochemical studies.

    Ophthalmology new progress in 1999; 19:169-171

     3 WANG Zhi-chong, GE Jian, Jin-Tang Xu, Chen Qi. Corneal

    immunogenicity analysis of different organizations. Chinese Journal of Ophthalmology, 2002; 8 (9) :535-538

     4 Li DQ, Tseng SC. Three patterns of cytokine expression potentially involved in epithelial-fibroblast interactions of

    human ocular surface. J Cell Physiol, 1995; 163 (1) :61-79

     5 Friend J, Kinoshita S, Thoft RA, Eliason JA. Corneal epithelial cell cultures on stromal carriers. Invest

    Ophthalmol Vis Sci, 1982; 23 (1): 41-49

     6 Wu Jing, Jin-Tang Xu, Song-Bin Zhao. Cultured corneal

    epithelial transplantation experiments. Chinese Journal of Practical Ophthalmology, 2001; 19 (3) :178-179

     7 Amano S. Transplantation of cultured human corneal

    endothelial cells. Cornea, 2003; 22 (7) :66-74

     8 Zhang Chao, Jin Yan, Dan Hu, Nie Xin, Liu Yuan, LEI Juan. Xenogenic acellular cornea material biocompatibility studies. International Ophthalmology, 2005; 5 (2) :250-253 reposted

    elsewhere on the free Paper Download Center http://

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