Weeks In Organ Culture Research Articles

Overview of Short-Term and Intermediate-Term Corneal Storage Solution: Comparison of Clinical Outcomes and Need For Future Research.

Corneal donor tissue preservation techniques have incrementally improved since the introduction of McCarey-Kaufman storage solution from short-term storage to intermediate duration of storage with the advent of organ culture and Optisol GS storage solutions. Improved understanding of the corneal endothelial cell physiology has helped in designing newer storage solutions, such as the Life 4C and Cornea Cold. The incorporation of antibiotics, ATP precursors, minerals, and vitamins has improved the viability of tissues. In addition, these modifications to the newer storage solutions have increased the endothelial longevity and metabolic activity. Despite these advances, the duration of tissue storage has largely been restricted to 2 weeks in Optisol GS and 4 weeks in organ culture. The role and cost-effectiveness of antifungal supplementation and the need for improved epithelial preservation are additional areas that need to be explored. This review intends to summarize the efficacy and viability of donor corneas in different tissue storage solution and compare clinical outcomes while providing an insight into the challenges in developing newer methods of corneal preservation.

Early wound healing of laser in situ keratomileusis–like flaps after treatment with human corneal stromal stem cells

PurposeTo use a well-established organ culture model to investigate the effects of corneal stromal stem cells on the optical and biomechanical properties of corneal wounds after laser in situ keratomileusis (LASIK)–like flap creation.SettingSchool of Optometry and Vision Sciences, Cardiff University, Cardiff, Wales, United Kingdom.DesignExperimental study.MethodsThe LASIK-like flaps were produced in sheep corneas. The flap beds were treated with corneal stromal stem cells and were then replaced and allowed to heal for different periods of up to 3 weeks in organ culture. The optical transmission of the cornea, the force required to detach the flap, and the presence of myofibroblasts near the flap bed were measured.ResultsCorneal stromal stem cell–treated flap beds were statistically significantly more transparent after 3 weeks in culture than the untreated controls. At 3 weeks, the mean force necessary to detach the flap was more than twice the force required for the respective control samples. Concurrently, there were 44% activated cells immediately below the flap margin of the controls compared with 29% in the same region of the corneal stromal stem cell–treated flaps.ConclusionsIn this system, the presence of corneal stromal stem cells at the wound margin significantly increased the adherence of LASIK-like flaps while maintaining corneal transparency. It is postulated that this is achieved by the deposition of extracellular connective tissue similar to that found in the normal cornea and by the paucity of activated keratocytes (myofibroblasts), which are known to scatter a significant amount of the incident light.Financial DisclosureNo author has a financial or proprietary interest in any material or method mentioned.

Split Cornea Transplantation: Relationship between Storage Time of Split Donor Tissue and Outcome

To analyze the relationship between storage time of split donor tissue and outcomes after deep anterior lamellar keratoplasty (DALK) and Descemet's membrane endothelial keratoplasty (DMEK). Retrospective analysis of a nonrandomized, consecutive, interventional case series. One hundred ten eyes with anterior stromal disease suitable for DALK and 110 eyes with endothelial disease suitable for DMEK underwent surgically successful split cornea transplantation combining both procedures within 7 days after splitting. Split donor storage times (splitting to grafting) and total storage times (death to grafting) were correlated with the 1-year functional and morphologic outcomes after DALK and DMEK surgery using a Spearman correlation coefficient and a Mann-Whitney U test. Best spectacle-corrected visual acuity (BSCVA), endothelial cell density, and complication rates within 12 months of follow-up. The mean split donor storage time was 35 ± 47 hours (range, 0-162 hours) after splitting for anterior donor grafts and 21 ± 40 hours (range, 0-158 hours) for posterior grafts. The mean total storage time was 352 ± 108 hours (range, 108-678 hours) for anterior lamellas and 339 ± 109 hours (range, 96-630 hours) for posterior lamellas. One year after DALK, the mean BSCVA was 20/30 (range, 20/50-20/20), endothelial cell loss was 8% (range, 2%-16%), and the complication rate (Descemet's folds, epitheliopathy, loose sutures) was 18%. One year after DMEK, the mean BSCVA was 20/25 (range, 20/40-20/16), endothelial cell loss was 41% (range, 17%-63%), and the complication rate (partial graft detachment) was 62%. For DALK and DMEK, no significant association was observed between split donor storage time as well as total storage time and BSCVA (P ≥ 0.409), endothelial cell loss (P≥0.236), or complication rate (P ≥ 0.647) within 1 year of follow-up. Anterior and posterior donor tissue may be stored safely for up to 1 week in organ culture before use in DALK and DMEK surgery. This simplifies the clinical feasibility of split cornea transplantation to reduce donor shortage and cost in corneal transplantation in the future. The author(s) have no proprietary or commercial interest in any materials discussed in this article.

The impact of distension pressure on acute endothelial cell loss and neointimal proliferation in saphenous vein grafts

We aimed to determine the extent of acute endothelial cell loss and neointimal proliferation in the long-term in saphenous vein grafts (SVGs) exposed to defined distension pressures. During routine competence testing of SVGs for coronary artery bypass grafting (CABG), blinded peak pressure measurements were performed in 10 patients. In an experimental set-up, distension pressure-related endothelial damage was studied in the SVGs of 20 patients. In a subgroup (n = 10), each patient's SVG was divided into segments and subjected to four constant pressures (50, 100, 150 and 300 mmHg) for 30 min each. In another subgroup (n = 10), SVGs were exposed to a short phase of high pressure (low pressure followed by 300 mmHg for 5 min). Acute endothelial cell loss was quantified by CD31-immunostaining. After 2 weeks of organ culture, the neointimal proliferation was evaluated using histomorphometry. Pressure-related damage was compared with damage at baseline (0 mmHg). During routine competence testing for CABG, we revealed a median peak pressure of 355 mmHg (range: 240-639 mmHg). In the experimental set-up, significant acute endothelial cell loss occurred at all tested distension pressures: at 50 mmHg, the median endothelial cell loss was 29% (range: 20-51%, P = 0.015), at 100 mmHg 54% (range: 37-69%, P < 0.001), at 150 mmHg 75% (range: 41-88%, P < 0.001), at 300 mmHg 91% (range: 63-100%, P < 0.001) and at short high-pressure exposure 65% (range: 49-82%, P < 0.001) in comparison with 20% (range: 0-44%) at baseline. Significant neointimal proliferation occurred when a distension pressure of 50 mmHg was exceeded: at 50 mmHg, median neointimal proliferation was 97 µm (range: 60-380 µm, P = 0.176), at 100 mmHg 168 µm (range: 100-600 µm, P = 0.001), at 150 mmHg 183 µm (range: 160-440 µm, P < 0.001) at 300 mmHg 347 µm (range: 190-590 µm, P < 0.001) and at short high-pressure exposure 130 µm (range: 60-410 µm, P = 0.02) in comparison with 90 µm (range: 60-170 µm) at baseline. In vitro exposure of SVGs to low distension pressure ranges causes significant acute endothelial cell loss and crucial long-term damage, namely neointimal proliferation.

Sterility control and long-term eye-bank storage of cultured human limbal epithelial cells for transplantation

Background/aims:To assess sterility of cultured human limbal epithelial cells (HLEC) and to investigate the viability, morphology and phenotype of cultured HLEC following 2 and 3 weeks of organ culture storage.Methods:HLEC...

Donor tissue preparation for Descemet membrane endothelial keratoplasty

To evaluate a technique for preparing a donor Descemet membrane carrying autologous endothelium for transplantation in Descemet membrane endothelial keratoplasty (DMEK). Netherlands Institute for Innovative Ocular Surgery, Rotterdam, The Netherlands. A 9.5 mm diameter DM carrying autologous endothelium was stripped from 10 corneoscleral rims that had been organ cultured for 1 week. The endothelial cell density (ECD) was evaluated with light microscopy before and immediately after DM was stripped and during 4 additional weeks of organ culture. The mean ECD was 2701 cells/mm(2) +/- 302 (SD) before and 2719 +/- 322 cells/mm(2) immediately after DM was stripped and declined from 2604 +/- 352 cells/mm(2) after 1 week to 2190 +/- 768 cells/mm(2) after an additional 4 weeks of organ culture (n = 10). Typical "endothelial streaks," ie, linear cellular disruptions observed immediately after DM was stripped, showed complete regeneration after the second culture period. Descemet grafts for transplantation in DMEK procedures can be surgically prepared from organ-cultured corneal rims and stored for an additional 3 weeks with acceptable endothelial cell loss. Because the donor tissue can be dissected directly from organ-cultured corneoscleral rims, donor preparation for DMEK can be readily accessible to most corneal surgeons.

005 Analyses histologiques et ultra structurales des cornées humaines conservées en Corneamax et déturgées durant 48 heures dans les poloxamers

Poloxamers (P) are various synthetic block copolymers widely used in pharmaceutic industry. We previously described the efficiency of P188, 237, 338 and 407 to deswell human corneas at the end of organ culture (OC) and their ability to reduce the endothelial cell loss compared to 5% T500 Dextran in CorneaMax (with 2% foetal calf serum). We aimed to further assess the cellular effects of 48 hours incubation in P using histology and electron microscopy (EM). The P were dissolved in CMax (Eurobio, Les Ulis, France) at 350 mOsmol/L. Five paired human corneas with more than 2000 endothelial cells/mm2 used for each poloxamer. After 3 weeks of OC in CMax, one cornea was incubated for 48 hours in CMax + poloxamer, and the mate cornea in CorneaJet (CMax+ 5% T500 Dextran). Controls: fresh corneal button from keratoconus and lattice dystrophy (for normal unstored endothelium) and organ cultured corneas without deswelling. Corneas were divided into 3 parts for cross sectional histology (epithelial thickness measurement and counting of keratocytes), transmission EM of the posterior stroma and endothelium (cell morphology and interfibrillar spaces measurement) and scanning EM of the endothelium (morphology). Cross sectional histology showed differences in epithelial thickness. For both types of macromolecules (poloxamers and dextran) TEM revealed cytoplasmic vacuoles in endothelial cells and keratocytes and SEM confirmed the integrity of endothelial monolayer. Histological and ultrastructural findings, along with capacity of thinning, transparency improvement and endothelial cell preservation will allow selecting poloxamers to be added to CorneaMax for a future clinical application. This complementary validation using light and electron microscopy reasonably confirms the promising use of Poloxamer to replace T500 Dextran to deswell corneas during organ culture.

Experimental Study of the Survival of Metastatic Cancer Cells in Corneal Organ Culture

Transmission of donor malignancy to the recipient could be one of the most disastrous complications of corneal grafting. Because of the scarcity of donor tissue and the lack of sufficient scientific evidence, the harvest of donor tissues from deaths due to systemic malignancy is permitted. This study was conducted to investigate the possible transmission of donor metastatic disease via corneal tissue preserved in organ culture (OC) conditions. The viability of four frequent human cancer cell lines (lung, breast, skin, and colon) was studied in OC. Various inoculums of cancer cells labeled with the membrane marker PKH67 were seeded on donor corneas and preserved in OC, followed by cell-tracking studies, histopathology, and immunohistochemistry. HLA matching of the dissected Descemet's membrane (DM) of preserved corneas was conducted, to demonstrate cell adherence. Primary cell culture was performed to confirm the viability of adherent tumor cells. Viability tests showed a poor but persistent survival of cancer cells after 2 weeks in OC. Cell tracking, histopathology, and immunohistochemistry demonstrated cancer cell adherence to donor endothelium. HLA typing of the DM of preserved corneas revealed the presence of cancer cell alleles. Primary culture of the DM showed cell proliferation that was identical with the original cancer cell line, according to HLA studies. The findings demonstrate that under laboratory conditions, metastatic cancer cells adhere to donor corneal tissue, survive, and retain proliferative capacity during storage in OC. Cell lines differ in their viability potential, as well as the pattern of adherence to donor endothelium. Further in vivo experimentation in laboratory animals is need to determine the safety of such harvests.

Survival of frozen-thawed human ovarian fetal follicles in long-term organ culture

Ovarian follicles obtained from second and third-trimester human fetuses survived 4 weeks in organ culture and secreted 17-β estradiol (E 2).

Delivery of Genes and Fluorescent Dyes into Cells of the Intact Lens by Particle Bombardment

The authors report the use of a particle bombardment technique to deliver exogenous genes and fluorescent dyes into living fibre cells in the intact lens. Gold particles were coated with plasmid DNA encoding green fluorescent protein (GFP) or the lipophilic fluorescent probes DiI and DiO. The particles were introduced into embryonic chicken or neonatal mouse lenses using a pressurized helium charge. A significant fraction of particles penetrated the capsule and came to rest in the superficial lens cortex. Over the range tested, varying the particle size or the pressure of the helium charge had little effect on the final distribution of particles within the lens. Particle bombardment was used initially to deliver DiI and DiO into the lens. Within a few hours, bombarded lenses exhibited multicolored membranous labelling of individual, elongating fibre cells. The particle bombardment technique was also used to introduce a plasmid encoding GFP. After overnight incubation, many fibre cells in the bow region expressed GFP. On close examination by confocal reflectance microscopy, each expressing cell was found to contain a gold particle lodged in its nucleus. The authors examined the fate of GFP-expressing fibre cells over a period of 1 week in organ culture. In the embryonic chicken lens, transfected fibres showed modest (approximately two-fold) elongation. In contrast, GFP-expressing mouse lens fibres underwent dramatic elongation, reaching the anterior and posterior sutures after 7 days in culture. These species differences may reflect the fact that mitosis continued at a near normal rate in the cultured mouse lens but declined precipitously in the cultured chicken lens. These results suggest that particle bombardment, in conjunction with appropriate cell culture conditions, may prove useful in visualizing the behaviour of differentiating fibre cells in the living intact lens in vitro.

The dispersed cell culture as model for functional studies of the subcommissural organ: preparation and characterization of the culture system

The subcommissural organ (SCO) is an enigmatic secretory gland of the brain, which is believed to be derived from ependymal (glial) precursor cells. We here developed a dispersed cell culture system of the bovine SCO as an approach to functional analyses of this brain gland. Tissue of the bovine SCO obtained from the slaughterhouse was papain dissociated either directly after dissection or after preparation of SCO explants. The latter had been maintained for 4–6 weeks in organ culture. The dispersed cells were cultured for up to 14 days and continuously tested for their secretory state by immunostaining of their secretory product. With respect to the morphology of the SCO cells (shape, processes, nucleus), no difference was found between the culture of freshly dissociated SCOs and that of dissociated SCO explants. In all cases, the dissociation caused a dedifferentiation; typical elongated cells were formed increasingly after 1 day of culture. Thereafter, only the cellular size increased, whereas the shape and the viability of the cells remained unchanged. Proliferating SCO cells were never observed. The culture obtained from fresh SCO tissue contained more glia cells and fibrocytes than the culture prepared from SCO explants. The proliferation of glia cells and fibrocytes was suppressed by blocking the mitotic activity with cytosine-β- d-arabino furanoside (CAF). The cytophysiological features of the cultured dispersed cells of both origins did not differ as demonstrated by classical histology, by immunocytochemistry for the secretory products of the SCO, by the characteristics of calcium influx into the cytoplasm ([Ca 2+] i ) and cyclic adenosine monophosphate (cAMP) after stimulation with adenosine-5-triphosphate, substance P or serotonin, and by the activation of the transcription factor cAMP-responsive element-binding protein. Because of the maintenance of their viability, their capacity to release the secretory product into the culture medium, their receptive capacity, and their signal transduction pathways, we conclude that the dispersed cell culture system, especially that obtained from SCO explants, represents an appropriate and useful model for functional studies of the mammalian SCO.

Transplantation of cultured adult human or porcine corneal endothelial cells onto human recipients in vitro. Part II: Evaluation in the scanning electron microscope.

To evaluate the morphology of endothelial monolayers, which have been regrafted onto the denuded Descemet's membrane, with scanning electron microscopy (SEM). Material derived from each of the experimental groups described in part I of this investigation was evaluated in the current study. Recipient corneas, denuded of their native endothelium by mechanical, chemical, or physical debridement, were examined to assess the effectiveness of each technique in killing and removing cells. Porcine or human donor corneal endothelial cells maintained in monolayer culture for up to 10 passages then were seeded onto the denuded Descemet's membranes of recipients in the absence or presence of fibroblast growth factor (FGF). The monolayers thereby established were examined in the SEM, and the morphologic status of individual cells compared with that manifested in normal human donor corneas maintained for 4 weeks in organ culture (reference control). Isolated and cultured human keratocytes regrafted onto the denuded Descemet's membranes of recipient corneas served as nonendothelial control specimens. Tissue was processed for examination in the SEM according to standard techniques. Each of the three methods used to strip recipient corneas of their native endothelium was effective and elicited no gross structural damage to Descemet's membrane. Some small focal defects within this latter layer were, however, observed, these being encountered at higher frequency after mechanical debridement than after chemical or physical stripping. Porcine or human endothelial cells seeded onto the denuded Descemet's membranes of recipient corneas formed stable monolayers. The morphologic status of regrafted cells corresponded to that manifested in monolayer cultures before seeding, porcine ones always being more differentiated than their human counterparts. Poorly differentiated human endothelial cells had a slender, elongated, fibroblast-like appearance, whereas more highly differentiated ones manifested broad, flat, polygonal profiles. Monolayers covered the entire corneal surface and impinged to a variable degree onto the trabecular meshwork, at which juncture cells always assumed a less well-differentiated morphology. FGF consistently effected an increase in differentiation status, and as this became augmented, the capacity of monolayers to violate the corneal-trabecular meshwork border was correspondingly repressed. Seeded keratocytes formed dense, multilayered sheaths, resembling retrocorneal membranes, across the entire corneal surface, trabecular meshwork, and iris root. The surface characteristics of the constituent cells were quite distinct from those manifested by endothelial cells, even the least well-differentiated ones. Regrafting of human corneal endothelial cells onto the denuded Descemet's membranes of recipients resulted in the formation of stable monolayers. Because the morphologic status of seeded cells closely mimicked that manifested in monolayer cultures before transplantation, it may be anticipated that efforts to refine and optimize culturing conditions would yield improvements in this parameter after regrafting. If these expectations can be realized, then the possibility of successfully establishing a "new" and functional endothelium on recipient corneas destined for clinical grafting may well be brought to fruition in the not-too-distant future.

Correlation between glucose and lactate concentrations in the human cornea and in organ culture medium

Metabolic changes of the human donor cornea during organ-culture are not at all reflected by the endothelium. Therefore metabolic investigations have become of increasing interest. It was the aim of this study to determine the correlation between glucose and lactate in storage medium and within the cornea itself and to find thereby an additional parameter for glucose metabolism during organ-culture. Glucose and lactate were examined in 166 organ-culture medium samples as well as in 106 human corneas by enzymatical optical methods. Investigations were carried out after 1, 7, 15, 21 and 28 days of organ-culture. Glucose consumption was highest during the first two weeks of organ-culture. Glucose concentrations showed a good linear correlation between medium samples and the cornea (r = 0.923). The correlation coefficient for lactate was worse (r = 0.733). Glucose and lactate levels in the organ-culture medium can be used as a marker for glucose metabolism in the cornea.

Impact of Growth Factors on Morphometric Corneal Endothelial Cell Parameters and Cell Density in Culture-Preserved Human Corneas

Donor corneas can be preserved for < or = 4 weeks in organ culture (31 degrees C) by using modified minimal essential medium (MEM). About one fifth of them have to be discarded, however, as disintegration of the endothelial cell monolayer-enhanced polymegethism, cell loss-occurs. The objective of this study was to investigate whether addition of insulin, dextran, and epidermal growth factor (EGF) makes corneal endothelial cells more viable, stable, and homogeneous. Sixteen paired human donor corneas were cocultured in media supplemented with EGF and insulin-like growth factor (IGF) or in conventional modified MEM for 4 weeks. Endothelial parameters were evaluated at the outset and at days 7, 14, 21, and 28 of culture by using an automated digital image-analysis system. No significant differences were observed in the first 2 weeks of culture. Beginning with day 14, however, stabilization of endothelial cell patterns was evident for corneas cultured in supplemented culture media. Our data indicate that the addition of growth factors to culture media might increase the percentage of corneas available for transplantation and would also allow a significantly longer period of preservation.

Corneal Grafting of Donor Tissue Preserved for Longer than 4 Weeks in Organ-Culture Medium

In vitro viability of the endothelium of corneas stored in organ-culture medium is longer than in other storage techniques. This article reports on the clinical results of 20 penetrating keratoplasties performed using corneas stored for > 4 weeks in organ culture. Seventeen of the procedures were elective and three were emergencies. Mean preservation time was 35.6 +/- 7 days (mean +/- SD) and mean follow-up was 16.2 months. During preservation, an endothelial cell loss of 10% was noted. Seventeen grafts remained transparent, and three failed between 3 and 24 months postoperatively. Mean endothelial cell density was 2,153 +/- 372/mm2 at 4 months and 1,854 +/- 390/mm2 at 12 months. Central pachymetry was 548 +/- 53 microns at 4 months and 528 +/- 57 microns at 12 months after surgery. Corneas stored for up to 48 days in organ culture can be used in elective as well as in emergency procedures.

Organization of tracheal epithelium in the cartilaginous portion of adult rabbit and its persistence in organ culture

The rabbit trachea provides a model system to test the physiological responses of the airways to various agents. Since three-dimensional organization may affect responses of an organ, an organ culture model was established in serum free medium. Tracheas were fixed in situ, at steps in the preparation of organ cultures, and after one day to three weeks in organ culture. Samples were examined by light microscopy and scanning and transmission electron microscopy for surface morphology, distribution of cell types, and characteristics of the epithelial cell layer. The normal tracheal mucosa was discovered to consist of extensive circumferential folds in the cartilaginous portion, which were enhanced upon isolation of the trachea from the animal. The folds consisted principally of differences in epithelial cell height rather than folding of the lamina propria. Enhancement of folding upon removal of the trachea coincided with increased secretion by Clara and possibly mucous cells. In organ culture, epithelial cell height initially increased, producing tall folds and cell types were retained in normal proportions. After prolonged culture, cilia were lost but glandular secretion continued. Changes in the arrangement of basal cells and secretory activity in isolated trachea and in culture may give insight into the functional significance of the epithelial folds.

Morphological and Physiological Development of Vestibular Hair Cells in the Organ-Cultured Otocyst of the Chick

The inner ear of the embryonic chick forms an oval-shaped sac or otocyst, on Embryonic Day 3, which contains presumptive sensory and support cells. After 3 weeks in organ culture the otocyst had sensory epithelia with an average of 325 ± 41 hair cells. Using light and transmission electron microscopy most of these cells were identified morphologically as type II vestibular hair cells. Whole-cell tight-seal recordings, using potassium chloride-filled micropipetes, showed that mature cultured hair cells had four different types of K currents. These included: a voltage-gated delayed rectifier K + current ( I K), an inactivating K + current ( I A), a calcium-dependent K + current ( I K(Ca)), and a K + inward rectifier ( I IR). These currents were similar to those recorded from cristae ampullares cells isolated from 2- to 3-week-old posthatched chicks. We also determined the timing of K + current acquisition in vitro. Initially, recordings showed that cells isolated from Embryonic Day 3 otocysts had no voltage-dependent outward currents at physiological membrane potentials. Eventually, K + currents were acquired in the order of: I K and I IR after 9 days, I A after 12 days, and I K(Ca) after 17 days in vitro . In addition, recordings using cesium chloride-filled micropipetes showed that there were two types of inward currents that were elicited in response to membrane depolarizations. These two currents included a rapidly activating, noninactivating Ca 2+ current and a tetredotoxin-sensitive Na + current. Both currents were elicited in hair cells grown in vitro for 13 days. Although I Na was previously unreported in avians, both I Na and I Ca were also represented in hair cells isolated from the cristae ampullares of the posthatched chick. These results indicate that hair cells can acquire voltage-gated currents in vitro which are qualitatively similar to ionic currents found in crista ampullaris cells that differentiate in vivo. Thus, this organ culture system provides a means to study regulation of ionic currents in developing hair cells.

Healing of Rabbit Corneal Alkali Wounds In Vitro

The repair process of rabbit corneal alkali wounds was examined for 3 weeks in organ culture. The initial wound was inflicted by applying a round filter paper, 5.5 mm in diameter, soaked in 1 N NaOH, for 60 s on the central cornea--the same procedure as used in previous in vivo studies. Epithelial and endothelial healing were determined morphometrically by utilizing a standardized photographic procedure following alizarin red/trypan blue staining. A one-phase healing was discerned both in the epithelium and in the endothelium. The epithelial resurfacing of the wound was completed by 5 days and then remained intact. However, the endothelial defect did not heal completely during the observation period of 21 days. The defect area was, however, consistently reduced in size during the first four days. The present results were compared with previous in vivo findings in rabbit eyes. The advantages of using an in vitro model for initial therapeutic trials are described.

Morphology of pig retinal pigment epithelium maintained in organ culture.

Exoplants of porcine retinal pigment epithelium (RPE) attached to Bruch's membrane, choroid, and sclera were maintained in organ culture for up to four weeks. Four-millimeter round buttons of eye wall that contained RPE, choroid, and sclera were trephined from freshly enucleated pig eyes and incubated at 37 degrees C in Eagle's minimum essential medium with 10% fetal calf serum. The RPE cells remained as a monolayer for at least four weeks in organ culture, and individual RPE cells became taller and dome shaped. The RPE cells retained several prominent ultrastructural features, including apical microvilli, intracellular melanosomes and mitochondria, and intercellular tight junctions. Since the cellular substratum can exert important influences on cell behavior, the ability to maintain RPE cells attached to Bruch's membrane provides a new in vitro tool for studying the metabolic activity of this tissue and its response to external stimuli, including laser photocoagulation.

EXPERIMENTAL STUDY OF HUMAN GALL-BLADDER EPITHELIUM BY ORGAN CULTURE IN VITRO AND XENOTRANSPLANTATION TO NUDE MICE

This paper presents results of the long-term maintenance of human gall-bladder epithelia as xenografts following explant organ culture. The sequential changes in morphological appearance during the period of in vitro organ culture as well as in vivo xenografts were extensively studied. Surgically resected gall-bladder tissues (8 cases with cholecystolithiasis and one with cholecysto-choledocholithiasis) provided the materials used in this study. They were cut into small fragments and maintained as explant organ cultures according to the method of Resau et. al. After several weeks of organ culture, about half of the explants were xenotransplanted into nude mice. The xenografts recovered at particular intervals were studied by microscopy and histochemistry. The results were as follows.In explant tissues, sequential changes in morphology, which included degeneration, regeneration and proliferation of the epithelia, were observed during the course of in vitro culture. These findings were substantiated by the viable epithelia composed of flat, cuboidal and columnar cells. The morphological viability was well preserved during the initial three weeks. However, it began to deteriorate thereafter, and no viable epithelia were obtained after 7 weeks. In contrast to the morphological viability, lactate dehydrogenase activity in the explant media showed bi-phasic elevation. The first peak, which occurred within 24 hours, was remarkably high ; however, the second peak, at around 10 days, was relatively low. These findings in morphology and enzymology are compatible with the process generally observed after cell injury and subsequent regeneration.In the xenografted tissues, the formation of multiple cysts of various sizes was noted. These cysts and the surrounding tissue contained various kinds of morphological architecture including projection of proliferated epithelial cells in the lumen and sinus-like structures. The viability of the epithelial cells composing the inner surface of the cyst walls was excellent. These cells showed various stages of morphological differentiation (flat, cuboidal and tall columnar cells) depending on how long the xenograft was maintained in vivo. In addition, acid muco-substance or sulfomucin, which was evidenced by positive staining with periodic acid-Schiff-alcian-blue and high-iron-diamine-alcian-blue, was observed in the tall columnar epithelial cells. These findings indicate functional differentiation in these cells.These results may extend the knowledge of human gall-bladder organ culture, and indicate that the method employed in the present study is potentially applicable to the study of human gall-bladder carcinogenesis in an experimental model.