Wound healing after excision of mantle tissue from the Akoya pearl oyster, Pinctada fucata

Pearl oysters are usually sacrificed to donate mantle tissue for pearl production. However, if oysters are anaesthetized, they are able to survive mantle excision and regenerate this tissue. Mantle excision causes a large wound and severs the pallial artery that necessitates rapid wound repair to avoid death by bleeding. This study was undertaken to assess the wound healing process in the mantle of the Akoya pearl oyster, Pinctada fucata, following mantle excision. Forty-seven P. fucata were relaxed with 2.5 mL L(-1) propylene phenoxetol before mantle tissue was excised. Oysters were relaxed and sacrificed 1, 3, 6, 12, 25, 36, 48, 66, 80 and 105 h after excision to assess mantle healing using histological techniques. Muscular contraction that effectively reduced the size of the wound was observed within 1 h after mantle excision. Accumulation of haemocytes and connective tissue occurred 3-6 h after excision and wound plugging was achieved within 6 h of excision. Proliferation of epithelial cells to cover the wound site was observed within the first 25 h after mantle excision and growth of connective tissue and formation of the pallial artery were observed within 105 h after mantle excision.     

Monolayer formation and DNA synthesis of the outer epithelial cells from pearl oyster mantle in coculture with amebocytes

Summary In vitro experiments were conducted to clarify the involvement of the epithelium-amebocyte interaction in epithelial regeneration of bivalves. The outer epithelia of the pallial mantle of the pearl oyster, Pinctada fucata martensii, were separated in cell sheets from the inner connective tissue layers by digestion with Dispase. Clumps of the separated mantle epithelia were inoculated onto the amebocyte layers prepared on the bottom of culture dishes and maintained at 20° C in 5% CO₂:95% air for 1 wk. Balanced salt solution with 0.03% (wt/vol) glucose was used as a culture medium. The epithelial cells adhered to the amebocyte layers within 24 h, changed their shape from cuboidal to squamous, and migrated and formed monolayer sheets within 3 d. Electron microscopy confirmed maintenance of epithelial polarity and cell to cell junction in the sheets; 6 d after the inoculation, 5-bromo-2′-deoxyuridine was added to the culture at 30 μM. After labeling for 24 h, the cultures were fixed and stained with anti 5-bromo-2′-deoxyuridine antibody. Cells with immunoreactive nuclei were clearly observed in the epithelial cell sheets, indicating active DNA synthesis in the epithelial sheets. Thus, cocultured with amebocytes, the outer epithelial cells from pallial mantle tissue formed a monolayer sheet and started DNA synthesis. The morphological features of the mantle outer epithelial cells are analogous to those described for the in vivo cutaneous wound healing process, suggesting that the epithelium-amebocyte interaction is important in the regeneration of epithelium in bivalves.     

Characterization of Calcium Deposition and Shell Matrix Protein Secretion in Primary Mantle Tissue Culture from the Marine Pearl Oyster <Emphasis Type="Italic">Pinctada fucata</Emphasis>

In this study, we established and characterized a long-term primary mantle tissue culture from the marine pearl oyster Pinctada fucata for in vitro investigation of nacre biomineralization. In this culture system, the viability of mantle tissue cells lasted up to 2 months. The tissue cells were demonstrated to express nacre matrix proteins by RT-PCR, and a soluble shell matrix protein, nacrein, was detected in the culture medium by Western blot analysis. On the other hand, 15 days after initiating culture, a large amount of calcium deposits with major elements, including calcium, carbon, and oxygen, were generated in the mantle explants and cell outgrowth area. The quantity and size of calcium deposits increased with the prolonged cultivation, and their location and nanogranular structure suggested their biogenic origin. These calcium deposits specifically appeared in mantle tissue cultures, but not in heart tissue cultures. Taken together, these results demonstrate that the mantle tissue culture functions similarly to mantle cells in vivo. This study provides a reliable approach for the further investigation on nacre biomineralization at the cellular level.     

淡水贝类贝壳多层构造形成研究

对几种淡水贝（包括蚌、螺）进行形态及组织学观察，并通过实验方法重现贝壳三种物质，即：角质、棱柱质、珍珠质的生成过程，结果表明：外套膜外表皮细胞是由相同类型细胞组成，这些相同细胞在不同的作用条件下形成贝壳多层构造。     

Gene Expression Patterns in the Outer Mantle Epithelial Cells Associated with Pearl Sac Formation

For pearl culture, nucleus and mantle grafts are implanted into the gonad of the host oyster. The epithelial cells of the implanted mantle graft elongate and surround the nucleus, and a pearl sac is formed. Shell matrix proteins secreted by the pearl sac play an important role in pearl formation. We studied the gene expression patterns of six shell matrix proteins (msi60, n16, nacrein, msi31, prismalin-14, and aspein) in the epithelial cells associated with pearl sac formation. There were differences in the expression patterns of the six genes in the epithelial cells, and the relative expression levels for msi60 and aspein differed between the mantle graft and pearl sac (48 days after implantation). Therefore, the gene expression patterns of the epithelial cells were genetically undetermined, and changed between before and after pearl sac formation. The gene expression patterns of the epithelial cells of the pearl sac may be regulated by the host oysters.     

圆背角无齿蚌血细胞在体内和体外损伤修复过程中的功能

以圆前角无齿蚌为材料,采用外套膜小片的异体移植与加入血细胞的体外培养,研究了血细胞在体风,唯物 修复及珍珠囊构建中的功能。结果表明人工育珠手术后的损伤修复中,血细胞有形成无颗粒细胞层修复伤口、６诱导表皮细胞迁移和再生并形成珍珠囊的功能,而在体外的组织培养中加入血细胞比不加入血细胞的对照组,能更快地诱导上皮细胞迁移和再生,在整个伤面形成２覆盖层,即形成类似珍珠囊结构。本文首次在国内、外报道了用细胞培     

Growth and differentiation of fetal rat small intestinal epithelium in tissue culture: Relationship to fetal age

Explants of small intestinal tissue have been cultured from fetal and young rats (from 13-day fetuses to 3-week-old rats). Growth of morphologically typical epithelial cells was obtained from explants of tissue from 14–20 day fetuses. Optimal growth was obtained using tissue from 17-day fetuses with outgrowth from the explant being observed 1-day after explant. Eighty per cent of explants developed epithelial growth by 11 days in culture. Initially, the epithelial outgrowth showed no morphological evidence of differentiation but after 5–10 days in culture differentiation into goblet or elongated cells with alkaline phosphatase activity occurred. Cells with brush borders and goblet cells were identified using electron microscopy. No differentiation occurred if the explant was removed even though growth continued.It was very difficult to culture tissue from fetuses older than 20 days' gestation, and when small intestine of 18–20-day fetuses was divided into two parts (proximal and distal) and cultured separately, growth of epithelial cells from explants of the proximal segment was less successful than that of the distal segment, indicating that the growth ability of these epithelial cells in vitro was closely related to tissue maturation in vivo. In contrast to the apparent relationship between fetal age and successful growth of intestinal epithelial cells, squamous epithelial cells of the esophagus could be grown from explants of 14-day fetus through newborn and 3-week-old rats.     

Primary Human Bronchial Epithelial Cells Grown from Explants

Human bronchial epithelial cells are needed for cell models of disease and to investigate the effect of excipients and pharmacologic agents on the function and structure of human epithelial cells. Here we describe in detail the method of growing bronchial epithelial cells from bronchial airway tissue that is harvested by the surgeon at the times of lung surgery (e.g. lung cancer or lung volume reduction surgery). With ethics approval and informed consent, the surgeon takes what is needed for pathology and provides us with a bronchial portion that is remote from the diseased areas. The tissue is then used as a source of explants that can be used for growing primary bronchial epithelial cells in culture. Bronchial segments about 0.5-1cm long and ≤1cm in diameter are rinsed with cold EBSS and excess parenchymal tissue is removed. Segments are cut open and minced into 2-3mm³ pieces of tissue. The pieces are used as a source of primary cells. After coating 100mm culture plates for 1-2 hr with a combination of collagen (30 μg/ml), fibronectin (10 μg/ml), and BSA (10 μg/ml), the plates are scratched in 4-5 areas and tissue pieces are placed in the scratched areas, then culture medium (DMEM/Ham F-12 with additives) suitable for epithelial cell growth is added and plates are placed in an incubator at 37°C in 5% CO₂ humidified air. The culture medium is changed every 3-4 days. The epithelial cells grow from the pieces forming about 1.5 cm diameter rings in 3-4 weeks. Explants can be re-used up to 6 times by moving them into new pre-coated plates. Cells are lifted using trypsin/EDTA, pooled, counted, and re-plated in T75 Cell Bind flasks to increase their numbers. T75 flasks seeded with 2-3 million cells grow to 80% confluence in 4 weeks. Expanded primary human epithelial cells can be cultured and allowed to differentiate on air-liquid interface. Methods described here provide an abundant source of human bronchial epithelial cells from freshly isolated tissues and allow for studying these cells as models of disease and for pharmacology and toxicology screening.Download video file.^{(144M, mp4)}     

Structure and metabolism of glycoproteins and glycosaminoglycans secreted by organ cultures of rabbit trachea.

1. When cultured in medium 199 in an atmosphere of O2+CO2 (95:5) rabbit tracheal explants retained their viability for up to 21 days. 2. The explants secreted into the culture medium three electrophoretically separable components which were eluted in the non-retarded fraction from Sephadex G-200. 3. Digestion with papain and fractionation with a LiCl gradient on DEAE-cellulose resulted in the separation of these components, which were identified as a sialic acid-rich glycoprotein of epithelial origin, and chondroitin sulphate and hyaluronic acid from sub-epithelial cartilage and connective tissue. 4. Incorporation of radioactive precursors ( D-[U-14-C]glucose, D-[1-14-C]glucosamine, D-[1-14-C]galactose and Na2-35SO4) into these secreted macromolecules was indicative of their active biosynthesis by the tracheal tissue.     

Molecular cloning and characterization of perlucin from the freshwater pearl mussel, Hyriopsis cumingii

Perlucin is an important functional protein that regulates shell and pearl formation. In this study, we cloned the perlucin gene from the freshwater pearl mussel Hyriopsis cumingii, designated as Hcperlucin. The full-length cDNA transcribed from the Hcperlucin gene was 1460 bp long, encoding a putative signal peptide of 20 amino acids and a mature protein of 141 amino acids. The mature Hcperlucin peptide contained six conserved cysteine residues and a carbohydrate recognition domain, similar to other members of the C-type lectin families. In addition, a “QPS” and an invariant “WND” motif near the C-terminal region were also found, which are extremely important for polysaccharide recognition and calcium binding of lectins. The mRNA of Hcperlucin was constitutively expressed in all tested H. cumingii tissues, with the highest expression levels observed in the mantle, adductor, gill and hemocytes. In situ hybridization was used to detect the presence of Hcperlucin mRNA in the mantle, and the result showed that the mRNA was specifically expressed in the epithelial cells of the dorsal mantle pallial, an area known to express genes involved in the biosynthesis of the nacreous layer of the shell. The significant Hcperlucin mRNA expression was detected on day 14 post shell damage and implantation, suggesting that the Hcperlucin might be an important gene in shell nacreous layer and pearl formation. The change of perlucin expression in pearl sac also confirmed that the mantle transplantation results in a new expression pattern of perlucin genes in pearl sac cells that are required for pearl biomineralization. These findings could help better understanding the function of perlucin in the shell and pearl formation.     

Reorganization of intermediate filament cytoskeleton in induced metanephric mesenchyme cells is independent of tubule morphogenesis

The metanephric mesenchyme becomes converted into epithelial tubules if cultured in transfilter contact with an inductor tissue. The expression of intermediate filaments (IFs), used as cell-type-specific markers has been studied in this model system for differentiation and organogenesis. In immunofluorescence microscopy of frozen sections, the undifferentiated cells of isolated metanephric mesenchymes uniformly showed IFs of vimentin type only. Also, when cultured as a monolayer, cells from the uninduced mesenchymes showed only vimentin filaments. In frozen sections of transfilter explants, epithelial tubules apparently negative for vimentin could be seen after 3 days in culture, but expression of cytokeratin could not be demonstrated in the developing tubules until the fourth day of culture. Sections of explants cultured further showed tubule cells with distinct fibrillar cytokeratin positivity. The appearance of cytokeratin in the explants was also demonstrated with immunoblotting experiments, using two different cytokeratin antibodies. Expression of IFs was further examined in monolayer cultures of metanephric mesenchymes which had been initially exposed to a short transfilter induction pulse. In these experiments, cytokeratin-positive cells could be demonstrated after a total of 4 days in culture. Double immunofluorescence experiments showed varying amounts of vimentin in the cytokeratin-positive cells: after 4 days in culture, most cytokeratin-positive cells still showed vimentin-positivity although often in a nonfibrillar form. During further culture, gradual disappearance of vimentin-specific fluorescence was observed in cytokeratin-positive cells. The results suggest that the vimentin-positive metanephric mesenchyme cells lose their fibrillar vimentin organization upon induction that leads to kidney tubule formation. This change may be essential for the transformation from an undifferentiated mesenchymal cell into a specialized epithelial cell. Cytokeratin filaments, regarded as a marker for epithelial cells, seem to appear simultaneously with or soon after the change in vimentin organization. These changes in IF expression also occur in monolayer cultures of mesenchyme cells initially exposed to a short transfilter induction pulse. This suggests that epithelial differentiation, as revealed by the emergence of cytokeratin positivity, may occur even in the absence of a clear morphological differentiation and three-dimensional organization of the cells.     

In vitro culture of epithelial cells from the caput, corpus, and cauda epididymis of Sus domesticus

This work describes a protocol to culture epididymal epithelial cells from the caput, corpus, and cauda regions of Sus domesticus. Epididymal epithelial fragments were obtained by dissection and enzymatic digestion with collagenase. About 30 epididymal fragments from each epididymal region were cultured in 24-well culture plates with supplemented RPMI-1640 medium at 37 degrees C, 5% CO2 in air, and 100% humidity. A confluent monolayer of polygonal and tightly packed epithelioid cells from the three epididymal regions was obtained after 12-16 days in culture and maintained in vitro for more than 60 days. The proportion of epididymal epithelial cells in these cultures was assessed by immunofluorescent staining for cytokeratins. Throughout the 2 months of culture, about 80% of the cells were cytokeratin-positive. Electron microscopy observations indicated that cultured cells from caput, corpus, and cauda epididymal regions were tightly adhered to each other by junctional complexes and that stereocilia were present in their apical membranes. Moreover, the presence of an extensive rough endoplasmic reticulum, Golgi apparatus and numerous vesicles in the cytoplasm suggested that cultured cells maintained secretory and absorptive activities. These results show that the epididymal epithelial cells in culture from S. domesticus retain some fundamental features that characterize the epididymal epithelium in the intact organ. This system might be a valuable tool for studying the mechanism of sperm maturation in vitro, including epididymal cell secretions and the analysis of regional differences.     

Primary cell culture from gill explants of rainbow trout

Primary cultures of gill cells were initiated from gill filament explants of rainbow trout, Oncorhynchus mykiss . The explants were cultured in Leibovitz l -15 medium with 5, 10 or 20% foetal calf serum (FCS) and l -glutamine. The attachment efficiency was serum-dependent though increased FCS concentration did not stimulate further outgrowth of cells. The explants produced cell outgrowth 24 h after attachment as a sheet of cells which exhibited characteristics of gill pavement epithelial cells as indicated by surface microridges revealed by scanning electron micrographs. There was high proliferation for the first 14 days then a stable plateau for 30 days followed by a decline phase from 45 days. Following removal of cells, the explants produced further cell outgrowth which was especially active at the proliferation phase (14 days). Removal of these cells caused the explants to produce a further proliferation of cells reaching confluence in 10–14 days. After the third cell removal cell outgrowth from explants showed migratory activity but did not develop to resemble gill epithelial cells. The use of gill explants to establish primary cultures of fish gill cells has advantages which include longevity of the culture and successive proliferations from explants which could provide a useful tool for the investigation of long-term processes in cellular biology and reduce the number of culture preparations.     

Rat renal papillary tissue explants survive and produce epithelial monolayers in culture media made hyperosmotic with sodium chloride and urea

The capacity of papillary cells to adapt to elevated osmotic concentrations is unusual among mammalian cells. This capacity was evaluated by using primary tissue culture. Viability and growth of cells in rat renal papillary tissue explants were assessed after culture in media adjusted with urea and sodium chloride to various osmotic concentrations between 300 and 1,500 mOsm/kg water. The survival of cells, including cells resembling those of the collecting ducts and the loop of Henle, was greatest in medium adjusted to 1,000 mOsm with equiosmolar amounts of the two solutes. At 1,500 mOsm only cuboidal tubular epithelium resembling collecting duct epithelial cells survived. In contrast, cells of cortical tissue survived and grew at 300 and 640 mOsm, but not at 1,000 mOsm or above. Epithelial monolayers appeared to proliferate from collecting ducts and spread over the surface of the explants as well as onto the glass surface in the culture dish. Epithelial growth of medullary tissue was most rapid at 300 mOsm and was slower at 700 and 1,000 mOsm. Monolayers did not form at 1,500 mOsm; however, epithelial overgrowth of explants did occur. Hydropenia in the donor animal did not significantly affect the viability or growth of cultured papillary tissue. Explants cultured for 5 days at 300 mOsm followed by a stepwise increase in medium osmolality to 1,100 or 1,500 mOsm and cultured for 3 more days showed low or no survival whereas explants cultured at 700 mOsm survived such increases. Explants cultured for 5 days at 1,500 mOsm survived and grew monolayers when lowered to 300 mOsm. Poor viability and no epithelial proliferation were observed in explants cultured in medium adjusted to 900 mOsm with either urea or sodium chloride alone, suggesting that a mixture of the two solutes in the extracellular space, as found in vivo, may be essential in achieving elevated osmolalities.     

Culture of outer epithelial cells from mantle tissue to study shell matrix protein secretion for biomineralization

Mantle tissue plays an important role in shell biomineralization by secreting matrix proteins for shell formation. However, the mechanism by which it regulates matrix protein secretion is poorly understood, largely because of the lack of cellular tools for in vitro study and techniques to evaluate matrix protein secretion. We have isolated the outer epithelial cells of the mantle of the pearl oyster, Pinctada fucata, and evaluated cellular metabolism by measuring the secretion of the matrix protein, nacrein. A novel sensitive sandwich enzyme-linked immunosorbent assay (ELISA) was established to quantify nacrein. Mantle explant culture was demonstrated to provide dissociated tissue cells with high viability. Single dissociated cell types from explant culture were separated by density in a discontinuous Percoll gradient. The outer epithelial cells were isolated from other cell types by their higher density and identified by immunolabeling and ultrastructure analysis. ELISA assays revealed that the outer epithelial cells retained the ability to secrete nacrein in vitro. Moreover, increased nacrein secretion resulted from an increased Ca(2+) concentration in the culture media of the outer epithelial cells, in a concentration-dependent manner. These results confirm that outer epithelial cell culture and the ELISA method are useful tools for studying the regulatory mechanisms of shell biomineralization.     

Organ culture of adult rat colonic mucosa on fibrin foam

A system for maintaining adult rat colonic mucosa in organ culture for up to 28 days is described. Distal colonic mucosa physically separated from the muscle layers was cultured at 37 degrees C on a substrate of human fibrin foam in HEPES- and bicarbonate-buffered Waymouth's MB 752/1 medium supplemented with 10% fetal bovine serum, L-glutamine, bovine albumin, ascorbic acid, hydrocortisone, insulin, and ferrous sulfate; the optimal atmostphere for culture was 95% O2 and 5% CO2. Viability of explants was demonstrated by tissue morphology with light microscopy, incorporation of [3H]thymidine and [3H]leucine into DNA and protein, [14C]glucosamine and [3H]fucose incorporation, and glycoprotein synthesis. Two days after initiation of culture, degeneration of surface and crypt cells was observed. Secreted mucosubstances covered the explants. Explants maintained in 95% O2 retained a variable number of glandular crypts with normal columnar epithelium for 14 to 21 days in culture. At 28 days, explants contained a single layer of cuboidal surface epithelium and a rare cryptlike gland.     

Development of an in vitro culture method for cells and tissues from the zebra mussel (<Emphasis Type="Italic">Dreissena polymorpha</Emphasis>)

Despite the successful transfer of mammalian in vitro techniques for use with fish and other vertebrates, little progress has been made in the area of invertebrate tissue culture. This paper describes the development of an in vitro technique for the culture of both cells in suspension and tissue explants from the gill, digestive gland and mantle of the zebra mussel (Dreissena polymorpha) and their successful maintenance in culture for up to 14 days. Cell suspensions from the gills and digestive gland were the most successful technique developed with viability >80% maintained for up to 8 days in culture, suitable for use in short term toxicity tests. Tissue explants from the mantle were also maintained in culture for up to 14 days. This paper describes the challenges involved in the development of a novel in vitro culture technique for aquatic invertebrates.     

培养条件对三角帆蚌外套膜离体上皮组织珍珠质分泌能力的影响（简报）

珍珠是由珍珠贝外套膜的上皮细胞受到外源刺激物刺激形成珍珠囊（pearl—sac）,并由珍珠囊产生的钙质分泌物．其分泌物逐渐包围刺激原．使之体积急剧增长而形成的,珍珠质（nacre）是由大于95％的碳酸钙晶体与约5％的角壳蛋白组成的生物矿化产物。因此珍珠贝的外套膜在珍珠形成中起着重要的作用。珍珠贝外套膜的体外培养已开展了一些初步的研究工作。