Isolation and cultivation of equine corneal keratocytes, fibroblasts and myofibroblasts

Objective  To establish an in vitro model for the investigation of equine corneal wound healing. To accomplish this goal, a protocol to isolate and culture equine corneal keratocytes, fibroblasts and myofibroblasts was developed.
Animal material  Equine corneal buttons were aseptically harvested from healthy research horses undergoing humane euthanasia for reasons unrelated to this study. Slit-lamp biomicroscopy was performed prior to euthanasia by a board-certified veterinary ophthalmologist to ensure that all samples were harvested from horses free of anterior segment disease.
Procedure  Equine corneal stroma was isolated using mechanical techniques and stromal sub-sections were then cultured. Customized media at different culture conditions was used to promote growth and differentiation of corneal stromal cells into keratocytes, fibroblasts and myofibroblasts.
Results  Cell culture techniques were successfully used to establish a method for the isolation and culture of equine corneal keratocytes, fibroblasts and myofibroblasts. Immunohistochemical staining for alpha-smooth muscle and F-actin was used to definitively differentiate the three cell types.
Conclusion  Equine corneal stromal keratocytes, fibroblasts and myofibroblasts can be predictably isolated and cultured in vitro using this protocol.     

In vivo confocal microscopy of the normal equine cornea and limbus

Objective  To describe morphologic features, pachymetry and endothelial cell density of the normal equine cornea and limbus by in vivo confocal microscopy.
Animals studied  Ten horses without ocular disease.
Procedure  The central and peripheral corneas were examined with a modified Heidelberg Retina Tomograph II and Rostock Cornea Module using a combination of automated and manual image acquisition modes. Thickness measurements of various corneal layers were performed and endothelial cell density determined.
Results  Images of the constituent cellular and noncellular elements of the corneal epithelium, stroma, endothelium, and limbus were acquired in all horses. Corneal stromal nerves, the subepithelial nerve plexus, and the sub-basal nerve plexus were visualized. Cells with an appearance characteristic of Langerhans cells and corneal stromal dendritic cells were consistently detected in the corneal basal epithelium and anterior stroma, respectively. Median central total corneal thickness was 835 μm (range 725–920 μm) and median central corneal epithelial thickness was 131 μm (range 115–141 μm). Median central endothelial cell density was 3002 cells per mm² (range 2473–3581 cells per mm²).
Conclusions  In vivo corneal confocal microscopy provides a noninvasive method of assessing normal equine corneal structure at the cellular level and is a precise technique for corneal sublayer pachymetry and cell density measurements. A resident population of presumed Langerhans cells and corneal stromal dendritic cells was detected in the normal equine cornea. The described techniques can be applied to diagnostic evaluation of corneal alternations associated with disease and have broad clinical and research applications in the horse.     

Effects of growth factors (EGF,PDGF-BB and TGF-beta 1) on cultured equine epithelial cells and keratocytes: implications for wound healing

Objective The physiologic mechanisms involving growth factors, including PDGF‐BB, EGF, and TGF‐β1, as potent mediators of fibroblasts and epithelial cells in corneal wound healing remain unknown. The goal of this study was to determine culture methods for equine epithelial cells and keratocytes and to investigate how exogenous growth factors influence proliferation of both cell types. Procedures Cell cultures were established from healthy corneas harvested from horses immediately following euthanasia and maintained using standard tissue culture protocols. To determine the effects of PDGF‐BB, EGF, TGF‐β1, keratocytes (1 × 10⁵/well) and epithelial cells (2 × 10⁵/well) were each cultured in 12 well plates and exposed separately to the growth factors. The cells were exposed to concentrations of EGF between 0 and 50 ng/mL; PDGF‐BB between 0 and 75 ng/mL; and TGF‐β1 between 0 and 10 ng/mL. Cell proliferation was measured using ³H‐thymidine assay and differences in growth determined using anova and Tukey's HSD test (P < 0.05). Results Epithelial cell and keratocyte cultures were successfully established. EGF maximally stimulated keratocyte and epithelial cells at 25 ng/mL and 5 ng/mL, respectively. PDGF‐BB maximally stimulated keratocytes and epithelial cells at 50 ng/mL and 5 ng/mL, respectively. TGF‐β1 inhibited keratocytes at 5 ng/mL and 10 ng/mL, and epithelial cells at 1 ng/mL and 2 ng/mL. Conclusions Methods were established to maintain epithelial cells and keratocytes in vitro. PDGF‐BB and EGF stimulate, while TGF‐β1 inhibits the proliferation of epithelial cells and keratocytes. These growth factors may play a role in maintenance and repair of the equine cornea.     

Comparative observation of freeze–thaw-induced damage in pig, rabbit, and human corneal stroma

Objective  Although variations exist between species with respect to outcomes after cryopreservation, little is known about the differences in the susceptibility of the corneal stroma to cryoinjury. We performed this study to investigate freeze–thaw-induced damage in keratocytes and collagen in rabbit, pig, and human corneas.
Animals studied  Rabbit, pig, and human.
Procedures  We prepared 250-μm-thick anterior stroma from rabbit, pig, and human corneas after scraping off the epithelium and endothelium. Each 250-μm-thick corneal stroma without epithelium was placed in a 50-mL tube, frozen with liquid N₂ for 15 min and taken out to thaw rapidly at 37 °C. This procedure of rapid freezing and thawing was repeated three times. Differences between the species with respect to cells and collagen structures were examined using hematoxylin–eosin (H&E) staining, terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) assay, and transmission electron microscopy (TEM). We orthotopically transplanted the pig and rabbit corneal transplants after the triple freeze–thaw cycle into rabbit eyes and evaluated graft survival.
Results  On gross examination, rabbit corneas became opaque after the triple freeze–thaw procedure, while pig and human corneas remained transparent. Histologically, keratocytes were apoptotic on TUNEL assay and TEM in rabbit, pig, and human corneas. Collagen fibrils were fragmented and the arrangement of collagen fibrils was severely disturbed in rabbit corneas on H&E staining and TEM; collagen was well preserved in pig and human corneas. Rabbit corneal stroma underwent autolysis after transplantation, whereas the pig corneal stroma remained clear for 1 month.
Conclusions  Our study showed that rabbit corneal stroma was more susceptible to freeze–thaw injury than pig and human corneas.     

Ultrastructural findings in feline corneal sequestra

OBJECTIVES: (1) To describe the ultrastructural features of corneal sequestra in cats; and (2) to enhance our understanding regarding the pathogenesis of feline corneal sequestration. METHODS: Nine corneal sequestra were harvested via keratectomy from globes of nine cats. The sequestra were routinely fixed then postfixed for high resolution light and transmission electron microscopy (HR-LM and TEM, respectively). The tissues were embedded in Epon/Araldite. Sections of 0.5-microm thickness were cut and stained with 1% toluidine blue in 1% sodium tetraborate solution for HR-LM. Ultrathin sections were collected on copper grids and stained with uranyl acetate and Sato's lead stain for TEM. Ultrathin sections were examined and the images were captured on an Advantage HR CCD camera using a Hitachi 7500 electron microscope operated at 80 kV. Two healthy corneas from two cats were harvested immediately following euthanasia. These corneal tissues (control samples) were processed in the same manner as the corneal sequestra for HR-LM and TEM. A portion of each sequestrum was also submitted for polymerase chain reaction (PCR) testing for infectious agents including feline herpesvirus-1 (FHV-1), Toxoplasma gondii, Chlamydophila felis and Mycoplasma spp. RESULTS: Ultrastructure of healthy corneal tissues revealed basal corneal epithelial cells aligned adjacent to a thin acellular layer similar to Bowman's layer with underlying tightly packed, regularly arranged, collagen fibrils oriented in different planes. Keratocytes were elongated and had long and irregularly shaped nuclei, and cytoplasm contained rough endoplasmic reticulum and abundant membrane-bound vesicles. In contrast, corneal sequestra contained varying amounts of an amorphous, electron-dense substance, continuous with intact basal epithelial basement membranes peripherally, and overlying corneal ulceration and loosely packed collagen fibrils. Remnants of necrotic keratocytes were seen in spaces between disarranged collagen layers. In all samples, occasional keratocytes exhibited morphology indicative of apoptosis including clumping and margination of chromatin, and shrunken cytoplasm. Varying degrees of inflammation were noted on HR-LM and TEM of affected corneas including peri- and intralesional neutrophils, lymphocytes, plasma cells, and macrophages. Corneal sequestra were FHV-1-positive (n = 3), FHV-1- and T. gondii-positive (n = 1), T. gondii-positive (n = 3), or negative for DNA of these infectious agents (n = 2) using PCR. All corneal sequestra were negative for DNA of Chlamydophila felis and Mycoplasma spp. using PCR. CONCLUSIONS: Apoptosis may play a role in the pathogenesis of feline corneal sequestration independent of the presence of DNA of these infectious organisms. Prospective clinical studies are warranted to further understand the significance of T. gondii in relation to feline corneal sequestration.     

Basic fibroblast growth factor stimulates epithelial cell growth and epithelial wound healing in canine corneas

Objective  To evaluate the effect of basic fibroblast growth factor (bFGF) on the proliferation of canine corneal epithelial cells and epithelial wound healing.
Animal studied  Canine corneal epithelial cells from the corneas of euthanized dogs and corneal epithelial wounds on one eye from each of 24 dogs.
Procedures  The proliferation of corneal epithelial cells in vitro was measured using the methylthiazolyl-tetrazolium (MTT) assay. A corneal wound on one eye of each dog was made with a corneal trephine (6 mm diameter). Four concentrations of bFGF, 0, 100, 500, and 1000 ng/mL, were applied to the affected eyes of dogs, t.i.d. Fluorescein staining was used to assess closure of the corneal epithelial wound.
Results  The addition of bFGF resulted in a significant increase in epithelial proliferation at 24 h after culture, except 1 ng/mL bFGF. Cells with all bFGF treatments proliferated significantly at 48 and 96 h compared to those in the non-bFGF group. bFGF at a concentration of 10 ng/mL promoted cell proliferation maximally. The wound healing rate in the bFGF-treated groups was greater than that in the control. All corneal wounds in bFGF-treated corneas closed by day 7, whereas two of six corneal wounds in the control showed poor healing. None of the eyes developed corneal clouding or neovascularization during the experiment.
Conclusions  Basic fibroblast growth factor accelerated the proliferation of canine epithelial cells and effectively promoted corneal epithelial wound healing.     

Isolation and characterization of equine microvascular endothelial cells in vitro

The use of cultured tissue has not yet become widespread in research involving equine disease, and this may be attributable in part to the scarcity of published reports concerning tissue culture methods for this species. We report here the isolation of equine microvascular endothelium (EMVE) from fresh omental tissue of horses and ponies. Fresh donor tissue was minced, subjected to collagenase digestion, and filtered. Cells were layered on 5% bovine serum albumin for gravity sedimentation, the bottom layer was collected, and the cells were plated onto fibronectin-coated flasks. Medium consisted of Dulbecco modified Eagle medium with 10% whole fetal bovine serum (wFBS) and 20 micrograms of endothelial cell growth supplement/ml. The EMVE grew readily in culture, had the cobblestone morphologic feature at confluence, stained positively for factor VIII-related antigen, and metabolized acetylated low-density lipoprotein. Fibroblast and smooth muscle cell contamination was minimal in primary cell cultures, which were successfully passed and maintained in culture for 3 to 5 serial passages, using various media and substrates. Preliminary studies were undertaken to determine optimal growth conditions with a range of variables: serum concentration, extracellular matrix components, and growth factors, Optimal conditions were achieved with a minimum of 10% wFBS, and with either fibronectin or laminin as extracellular matrix substrates. The EMVE grew adequately in Dulbecco modified Eagle medium plus 10% wFBS, and the added growth factors or serum supplements did not appear necessary for growth of EMVE.     

Cryopreservation of equine mononuclear cells for immunological studies

A rapid and simple technique for the cryopreservation and recovery of equine mononuclear cells was developed. Buffy-coat leukocytes were frozen in autologous plasma containing 10% DMSO and mononuclear cells were recovered by gradient sedimentation using a standard Ficoll-Hypaque purification procedure. The total numbers of mononuclear cells recovered from cryopreserved samples were 94%-82% of those recovered from fresh blood samples. The functional capabilities of the mononuclear cells from cryopreserved buffy coat preparations were compared with those of mononuclear cells from fresh samples by measuring the ability of cells to proliferate in response to mitogens and specific antigens. Cell-surface antigen expression was measured using monoclonal antibodies in conjunction with flow cytometric techniques and alloantisera in a complement mediated cytotoxicity assay. Cryopreserved mononuclear cells were capable of proliferating normally when stimulated with several mitogens, pokeweed mitogen, phytohemagglutinin and concanavalin A, and a single specific antigen preparation, equine influenza-2 (Equi-2) proteins. The maximum levels of proliferation induced by varying the concentrations of mitogens or the Equi-2 proteins were the same for both the fresh and cryopreserved cells. However, the cryopreserved cells usually required one more day in culture to attain maximum proliferation levels. Flow cytometric analysis of the samples demonstrated that the relative proportions of different lymphocyte populations were not altered by the cryopreservation step. Similarly, MHS alloantigen expression was not altered. The simplicity of the technique coupled with the retained functional properties allows for the cryopreservation of large numbers of leukocytes and the ability to assay various immune functions at a later time.     

黄姑鱼肠道上皮细胞体外分离培养及鉴定

本研究旨在建立黄姑鱼(Nibea albiflora)肠道上皮细胞的体外分离培养及鉴定方法,为海水鱼类肠道功能及其发病机制相关研究提供细胞模型.以黄姑鱼肠道组织为研究对象,采用胰蛋白酶(0.25％)、胶原蛋白酶(1 mg/mL)和透明质酸酶(0.16 mg/mL)联合消化法对肠道组织进行不同时间(0、20、40、60和...     

黑白花奶牛乳腺上皮细胞冻存方法的研究

试验研究了北方地区黑白花奶牛乳腺上皮细胞的最佳冻存方法。采用内蒙古地区健康黑白花奶牛的新鲜乳腺组织,将组织进行Ⅱ型胶原酶消化后原代培养,培养的细胞经5代纯化后将密度调整为1×106个/mL。采用6种处理方法进行冻存,各设3个重复。液氮冻存1个月后,分别将6种不同方法冻存的细胞复苏后测定细胞活性,包括冻存死亡率、贴壁细胞占活细胞的比率、凋亡率等,同时进行形态学观察。结果显示,处理1组与2、3、5组间冻存死亡率存在极显著差异（P〈0.01）,处理1、4、6组间冻存死亡率无显著差异（P〉0.05）,处理2、6组间冻存死亡率存在极显著差异（P〈0.01）;处理4组的贴壁率高于处理2、5、6组,存在极显著差异（P〈0.01）。形态学观察结果发现,处理1、4组细胞较其他组生长状态良好。因此,处理4可作为黑白花奶牛乳腺上皮细胞的最佳冻存方法。     

荷斯坦奶牛乳腺上皮细胞的分离培养

目的：建立荷斯坦奶牛乳腺上皮细胞的分离培养方法。方法：采用组织块种植法培养奶牛乳腺上皮细胞,利用胰蛋白酶差时消化法分离、纯化上皮细胞。结果：成功培养出奶牛乳腺上皮细胞,显微镜下观察,纯化的乳腺上皮细胞呈典型上皮细胞形态,细胞之间排列紧密,呈鹅卵石铺路样,形态均一,多角形的单层聚集。通过荧光免疫细胞染色方法对细胞骨架蛋白-角蛋白18进行鉴定,呈现阳性反应。乳腺上皮细胞增殖旺盛,经25次以上传代后长势仍然良好。结论：采用组织块种植法结合胰酶差时消化法成功获得纯化的奶牛乳腺上皮细胞。     

Corneal squamous cell carcinoma in a dog: a case report

Purpose:  To report a case of primary corneal squamous cell carcinoma (SCC) in an English Bulldog. In addition, immunohistochemistry of the corneal tissue mass was performed using a panel of antibodies. A prominent feature of the present case was the clinical history of chronic keratitis due to eyelid abnormalities.
Results:  No papillomavirus antigen was detected in section of normal or neoplastic corneal tissue. The corneal epithelial cells were positive for pancytokeratins AE1/AE3 and MNF116, and E-cadherin. The neoplastic cells in close proximity to the normal epithelial lining were positive for both pancytokeratins and E-cadherin with gradual loss of staining toward the center of the neoplastic mass. Rare neoplastic cells demonstrated positive staining for caspase 3 and a large number was strongly positive for GADD45 and p53.
Conclusion and discussion:  The observed loss of the various cytokeratins, the strong p53 expression, and low numbers of caspase 3 positive cells were suggestive that a p53 mutation may have caused this primary corneal SCC. Over-expression of the tumor-suppressor gene p53 is likely to be a consequence of ultraviolet radiation exposure. Two factors, however, may have played a role in the formation of this primary corneal SCC: chronic irritation of the corneal surface (microtrauma) and exposure to UV radiation.     

Reconstruction of corneal epithelium with cryopreserved corneal limbal stem cells in a rabbit model

The integrity and transparency of the cornea plays a key role in preserving vision. This paper reports a procedure to create an artificial sheet of corneal epithelium from cryopreserved limbal stem cells (LSCs) and to use this for corneal transplantation. Corneal LSCs were isolated from biopsy specimens of rabbit limbal lamellar and cryopreserved in liquid nitrogen at 2–4 passages. The cells were grown in culture medium for 12–14 days on top of a cell-free human amniotic membrane framed on a nitrocellulose sheet. The corneal epithelium generated was transplanted into the right eyes of 14 LSC deficient (LSCD) rabbits (seven experimental animals, seven controls) with corneal damage. The seven LSCD rabbits in the experimental group were transplanted with a corneal epithelial sheet generated from the cryopreserved corneal LSCs. Four LSCD rabbits were used as the vehicle control and were transplanted with a cell-free amniotic membrane, and the remaining three LSCD rabbits were negative controls without transplantation. Over a 2-month recovery period, 2/7 animals in the experimental group recovered completely, four recovered partially and one did not respond. In the control groups, three negative controls and three vehicle controls lost their vision completely, and one of the vehicle controls partially recovered transparency of the cornea Following treatment, corneal transparency of the experimental rabbits was significantly improved compared to controls (P < 0.05). The results indicated that cryopreserved corneal LSCs can repair damaged rabbit cornea, suggesting a possible new clinical approach to reconstruction of corneal epithelium.     

Establishing and functional testing of a canine corneal construct

Purpose  To provide a model to be used for in vitro studies on drug effects in dogs, this study was conducted to establish a protocol for the construction of a three-dimensional corneal construct. Primary canine corneal cells and a rabbit corneal epithelial (RCE) cell line were used in comparison.
Methods  The corneal construct was assembled step by step in membrane inserts of a six-well plate over a total of 5 weeks, including culture at the air–liquid interface to allow a differentiation of the epithelial cells. The constructs were studied histologically.
Single cell cultures of canine corneal cells as well as RCE cells were stimulated with lipopolysaccharides (LPS) and sodium dodecyl sulfate (SDS). Treatment with different concentrations of dexamethasone was used to test its effects on the cellular prostaglandin E₂ (PGE₂) production. The same experiments were repeated with the corneal constructs and the reactions compared. Expression of the glucocorticoid receptor (GR) in the constructs was studied using immunohistochemistry.
Results  A protocol for the construction of a vital corneal construct was established and the morphological similarity to the canine cornea in vivo shown. The GR protein was detected in all three cell types of the constructs. Stimulation with LPS and SDS led only in the corneal constructs to a significantly increased PGE₂ production, which could be reduced by dexamethasone.
Conclusions  The corneal construct is an interesting system to test drug effects on corneal cells. It allows studies on a cornea-like system including all three major cell types.     

Immunohistochemical Localization of Vascular Endothelial Growth Factor (VEGF) and its Two Receptors (Flt-I and KDR) in the Endometrium and Placenta of the Mare During the Oestrous Cycle and Pregnancy

Polyclonal antisera for vascular endothelial growth factor (VEGF) and its two main receptor molecules, VEGF-I (Flt) and VEGF-II (KDR), were used in a conventional immunocytochemical staining method to localize these angiogenic ligand molecules in the endometrium and placenta of the mare during the oestrous cycle and pregnancy. The anti-VEGF and anti-Flt sera both labelled the lumenal and glandular epithelia of the endometrium throughout the oestrous cycle and both the invasive trophoblast cells of the endometrial cups and the non-invasive trophoblast of the allantochorion in pregnancy. The anti-KDR serum likewise stained the maternal and foetal epithelial layers during the oestrous cycle and pregnancy and it also labelled fibroblast-like cells in the endometrial and allantoic stromas and the endothelium of foetal and maternal capillaries. The results demonstrated that constant supplies of the principal vasculogenic and angiogenic factor, VEGF, and its two major receptors, Flt and KDR, are available on both the maternal and foetal sides of the placental barrier throughout gestation in the mare. They are presumed to facilitate the continuing development of the extensive foetal and maternal capillary networks that are such prominent features within the microplacentomes of the diffuse, epitheliochorial equine placenta.     

Canine pre-iridal fibrovascular membranes: morphologic and immunohistochemical investigations

Objective  Pathologic intraocular neovascularization is a key component of many canine ophthalmic diseases such as uveitis, retinal detachment, intraocular neoplasms, and corneal perforation. The purpose of this study was to evaluate the structure of pre-iridal fibrovascular membranes (PIFMs) associated with several different disease processes and to identify specific factors associated with their development in the canine eye.
Procedure  This study examined 36 enucleated canine eyes with the diagnosis of PIFM and one of the following: lens-induced uveitis, retinal detachment, iridociliary adenoma, corneal perforation, severe hyphema, or vitreal gliovascular membranes (canine ocular gliovascular syndrome, COGS). Three histologic stains and six immunohistochemical stains were performed in all 36 PIFM eyes and four histologically normal eyes, including: hematoxylin and eosin, alcian blue periodic acid schiff (PAS), Masson's trichrome, platelet endothelial cell adhesion molecule-1 (CD31), smooth muscle actin, vimentin, laminin, vascular endothelial growth factor (VEGF), and cyclooxygenase-2 (COX-2).
Results  Pre-iridal fibrovascular membrane extracellular matrix staining was consistent with collagen and mucins in all cases and positive for laminin in most cases. All PIFMs contained CD31-positive vessels and predominantly lymphoplasmacytic inflammation. Both PIFM vessels and spindle cells were positive for laminin, vimentin, smooth muscle actin, VEGF, and COX-2. Secondary intraocular pathology and immunohistochemical staining of other intraocular structures are also reported.
Conclusions  Pre-iridal fibrovascular membrane morphology and immunohistochemical characteristics were similar across six canine disease processes, suggesting analogous pathophysiologic mechanisms. COX-2 and VEGF were identified using immunohistochemistry and may play a role in PIFM development.     

Primary cell culture and morphological characterization of canine dermal papilla cells and dermal fibroblasts

Skin biopsies were taken from female dogs, the primary hair follicles isolated and the dermal papilla dissected. After incubation in supplemented Amniomax complete C100 medium in 24-well culture plates, the dermal papilla cells (DPC) grew to confluence within 3 weeks. Thereafter, they were subcultivated every 7 days. Dermal fibroblast (DFB) cultures were established by explant culture of interfollicular dermis in serum-free medium, where they reached confluence in 10 days. They were subcultivated every 5 days. For immunohistochemistry, cells were grown on cover slips for 24 h, fixed and stained with antibodies against collagen IV and laminin. DPC showed an aggregative growth pattern and formation of pseudopapillae. Intensive staining for collagen IV and laminin could be observed until the sixth passage. DFB grew as branching, parallel lines and showed only weak staining for collagen IV and laminin.     

The effect of chondrocyte cryopreservation on cartilage engineering

Chondrocytes were collected from the stifle joints of four pigs to study the effect of cryopreservation on the chondrogenic potential of chondrocytes. Half of the cells were cryopreserved for 3months. Polyglycolic acid scaffolds were cultured with fresh or cryopreserved chondrocytes for 4weeks. Cell morphology and the quality of engineered tissue were evaluated by scanning electron microscopy, histopathology and biochemical methods. More cells attached to scaffolds at 48h when fresh chondrocytes were seeded. At 4weeks, the numbers of cells, DNA and collagen II were greater in constructs engineered by fresh cells. However, the collagen II/DNA ratio did not differ between the two groups. More matrix was identified on a scanning electron microscope and by histopathology in the fresh group. Cartilage engineered with cryopreserved chondrocytes may contain less matrix and fewer cells. These findings most likely resulted from a lack of cell attachment on the matrix secondary to cryopreservation. Future studies are needed to further evaluate the mechanism by which cryopreservation may affect chondrocyte attachment.     

鸡小肠上皮细胞的分离培养与鉴定

选择组织块法分离培养鸡小肠上皮细胞(intestinal epithelial cells,IEC),采用机械刮除法和相差消化－相差贴壁法纯化细胞,0.05%的Trypsin-EDTA对获得的IEC进行消化传代,免疫细胞化学法鉴定IEC。结果表明,组织块培养法可分离出活性较强的IEC,并获得纯化的上皮细胞;形态学和免疫细胞化学法检测显示获得的细胞表面抗原呈阳性,鉴定为IEC;纯化的IEC可在体外稳定传代。