Ultrastructure of mineralized nodules formed in rat bone marrow stromal cell culture in vitro.

Rat bone marrow stromal cells were cultured in vitro. At days 14-15 of culture, dense clusters of polygonal cells were formed, and they mineralized 2-3 days later. The cells resembling osteoblasts or young osteocytes were histologically observed to be embedded in mineralized or unmineralized extracellular matrices of the nodules. Next, these mineralized nodules were electron-microscopically examined. The osteoblastic cells associated with the nodules had a well-developed rough endoplasmic reticulum, an evident Golgi apparatus and some mitochondria as their intracellular organellae. Some lysosomes and microfilaments were also visible in the cytoplasms. Moreover, some cells protruded cell processes toward the neighboring cells through the extracellular matrix. The extracellular matrix consisted of numerous collagen fibrils which were striated with 60-70 nm axial periodicity and which was similar to bone tissue collagen. A large number of matrix vesicles were scattered among the collagen fibrils in the unmineralized area of the nodules. In contrast, in the mineralized area, numerous matrix vesicles at different stages of maturation and many calcified spherules were observed. That is the mineralization in this culture system was considered to be initiated in association with the matrix vesicles and to progress along the collagen fibrils. From these findings, it was confirmed by the present study that the mineralized nodules formed in this bone marrow stromal cell culture were ultrastructurally similar to bone and that the mineralization also proceeded by going through the normal calcification process. This culture system is considered to be available to study osteogenic differentiation and calcification mechanisms.     

A tissue culture system supporting cartilage cell differentiation, extracellular mineralization, and subsequent bone formation, using mouse condylar progenitor cells

Undifferentiated progenitor cells of mandibular condyles of neonatal mice were kept in a tissue culture system for up to 9 days. After 2 days in culture, new chondroblasts developed within the explants, whereas the peripheries of the latter were occupied by undifferentiated cells undergoing mitosis. By 4 days in culture, many of the cartilage cells showed signs of hypertrophy, while the matrix revealed positive reactivity for type II collagen and matrix metachromasia. The process of maturation of cartilage cells appeared to have reached its final stages after 6 days in culture, at a time when the initial loci of matrix mineralization could be easily identified. Concomitantly, peripheral areas bordering the cartilaginous core, as well as portions of the cartilage, reacted positively for type I collagen and fibronectin. Light microscopy examination showed signs of new bone formation after 9 days in culture. The extracellular matrix at the upper portion of the explant, facing the medium-air interface, reacted intensely with antibodies against type I collagen and fibronectin, but not with antibodies against type II collagen. Further, the newly formed osteoid was found to have undergone mineralization, thus forming an expanded layer of new bony tissue. A close spatial association was found between mature, mineralized cartilage and new bone. Hence, we hereby introduce a new in vitro system serving as an experimental model for studies related to the differentiation of progenitor cells into chondroblasts, which in turn promote ossification.     

Lung organoid culture

An organoid culture system for lung cells is described in which morphogenesis of lung histotypic structures and differentiation of both pneumocytes type II and mesenchyme occur. The principle of this technique is the culture of mouse fetal lung cells at high density on a membrane filter at the medium/air interface. In the course of cultivation, cell sorting-out, epithelial cell aggregation, formation of an alveolar-like lumen in the organoids and formation of a basal lamina occur. Epithelial differentiation culminates in the production of lamellar bodies, and the mesenchyme develops into mature connective tissue. Morphogenesis and differentiation depend on the stage of fetal development from which the lung cells were derived but appear independent of the formation of a basal lamina. Various drugs have been tested for their effects on morphogenesis and differentiation in this lung organoid culture: some of them inhibit differentiation or damage the mesenchyme, others stimulate surfactant production. Due to the quite complex morphogenetic and cellular events occurring in lung organoid culture, it may be an applicable tool for alternative in vitro screening methods.     

Alteration of gene expression levels during osteogenic induction of human adipose derived stem cells in long-term culture

Adipose tissue is a source of multipotent stem cells and it has the ability to differentiate into several types of cell lineages such as neuron cells, osteogenic and adipogenic cells. Most studies on human adipose-derived stem cells (ASCs) have been carried out at the early passages. For clinical usage, ASCs need to be expanded in vitro for a period of time to get sufficient cells for transplantation into patients. However, the impact of long-term culture on ASCs molecular characteristics has not been established yet. Several studies have also shown that osteogenic and adipogenic cells have the ability to switch pathways during in vitro culture as they share the same progenitor cells. This data is important to ensure their functionality and efficacy before being used clinically in the treatment of bone diseases. Therefore, we aim to investigate the effect of long-term culture on the adipogenic, stemness and osteogenic genes expression during osteogenic induction of ASCs. In this study, the molecular characteristics of ASCs during osteogenic induction in long-term culture was analysed by observing their morphological changes during induction, analysis of cell mineralization using Alizarin Red staining and gene expression changes using quantitative RT-PCR. Morphologically, cell mineralization at P20 was less compared to P5, P10 and P15. Adipogenesis was not observed as negative lipid droplets formation was recorded during induction. The quantitative PCR data showed that adipogenic genes expression e.g. LPL and AP2 decreased but PPAR-γ was increased after osteogenic induction in long-term culture. Most stemness genes decreased at P5 and P10 but showed no significant changes at P15 and P20. While most osteogenic genes increased after osteogenic induction at all passages. When compared among passages after induction, Runx showed a significant increased at P20 while BSP, OSP and ALP decreased at later passage (P15 and P20). During long-term culture, ASCs were only able to differentiate into immature osteogenic cells.     

Fresh and in vitro osteodifferentiated human amniotic membrane,alone or associated with an additional scaffold,does not induce ectopic bone formation in Balb/c mice

The human amniotic membrane (hAM) has been successfully used as a natural carrier containing amniotic mesenchymal stromal cells, epithelial cells and growth factors. It has a little or no immunogenicity, and possesses useful anti-microbial, anti-inflammatory, anti-fibrotic and analgesic properties. It has been used for many years in several indications for soft tissue repair. We previously reported that hAM represents a natural and preformed sheet containing highly potent stem cells, and could thus be used for bone repair. Indeed, native hAM possesses pre-osteoblastic potential that can easily be stimulated, even as far as mineralization, by means of in vitro osteogenic culture. However, cell culture induces damage to the tissue, as well as to cell phenotype and function. The aim of this study was to evaluate new bone formation by fresh and in vitro osteodifferentiated hAM, alone or associated with an additional scaffold presenting osteoinductive properties. Moreover, we also aimed to determine the effect of in vitro hAM pre-osteodifferentiation on its in vivo biocompatibility/tissue degradation. Results showed that neither fresh nor osteodifferentiated hAM induced ectopic bone formation, whether or not it was associated with the osteoinductive scaffold. Secondly, fresh and osteodifferentiated hAM presented similar in vivo tissue degradation, suggesting that in vitro hAM pre-osteodifferentiation did not influence its in vivo biocompatibility.     

Haemopoietic long-term bone marrow cultures from adult mice show osteogenic capacity in vitro on 3–dimensional collagen sponges

Abstract. Adult murine bone marrow cells, cultured under conditions for long-term haemopoietic marrow cultures, produce bone matrix proteins and mineralized tissue in vitro , but only after the adherent stromal cells were loaded on a 3-dimensional collagen sponge. Provided more than 8 × 10⁶ cells are loaded, mineralization as measured by ⁸⁵Sr uptake from the culture medium, occurred in this 3-dimensional configuration (3-D) within 6 days. In contrast if undisrupted marrow fragments (containing more than 10⁷ cells) are placed directly on a collagen sponge, then it requires more than 10 days before significant mineralization can similarly be detected. The 2-dimensional (2-D) long-term marrow culture system allows prior expansion of the stromal cells and some differentiation in an osteogenic direction within the adherent stromal layer. This is suggested by the presence of type I collagen and alkaline phos-phatase positive cells. However, synthesis of osteonectin and a bone specific protein, osteocalcin, as well as calcification are only observed in 3-D cultures. Electron microscopy demonstrated hydroxyapatite mineral on collagen fibres, osteoblast-like cells, fibroblasts, cells which accumulated lipids, and macrophages which were retained on the collagen matrices. Irradiation of confluent long-term bone marrow cultures, prior to their loading on the collagen sponge showed that haemopoietic stem cells are not necessary for the mineralization.     

Mineralization of adult mouse bone marrow in vitro

Murine adult bone marrow exhibits mineralizing capacity in vitro as is demonstrated by the new in vitro assay we report here. In less than 2 weeks after the onset of the cultures, mineralization is obtained in more than 80% of the marrow cultures. Moreover, morphological studies reveal that during incubation phenotypic changes related to osteogenic differentiation occur at the extracellular matrix as well within cell populations. Well banded collagen is synthesized. Matrix vesicles and needles of hydroxy-apatite crystals are observed via transmission electron microscopy. Osteoblast-like cells are present with membrane-associated alkaline phosphatase activity. The mineralization is specific for cultured bone marrow and is not observed in cultured spleen fragments as is shown via 85Sr uptake, calcein uptake and histomorphology. No inducing agent is added to the tissue culture medium except for 10% fetal calf serum, beta-glycerophosphate (10(-2) M) and ascorbic acid. However, the prerequisite for obtaining mineralization is the three-dimensional structure of the marrow in culture. The in vitro organ culture we developed may provide the opportunity to identify which marrow cells have osteogenic potential and to investigate the mechanisms triggering differentiation towards osteogenesis.     

Uptake of immunoglobulin and albumin by granulated metrial gland cells in vitro

Single cell suspensions of metrial gland tissue from rats at Day 14 of pregnancy were prepared for maintenance in vitro. During the first 2 days of culture IgG was detected in glycoprotein granule-containing granulated metrial gland (GMG) cells. Albumin was also detected in GMG cells at the same stages. The IgG and albumin were not detected during the next 4 days in culture. When metrial gland cells, maintained in vitro for 5 days, were incubated with rat serum for a further 24 h, IgG and albumin were detected in GMG cells. When similar cultures were incubated for 24 h with purified rat IgG or purified rat albumin, GMG cells were positive for IgG and albumin respectively. Albumin was not detected in GMG cells in wax sections of metrial gland tissue, although IgG has previously been demonstrated. The uptake of serum proteins by GMG cells in vitro has been clearly shown but the difference in IgG and albumin content of these cells in paraffin-wax sections indicates that the means by which IgG accumulates intracellularly may be different in vitro and in vivo.     

Mineralization of Adult Mouse Bone Marrow In Vitro

Abstract. Murine adult bone marrow exhibits mineralizing capacity in vitro as is demonstrated by the new in vitro assay we report here. In less than 2 weeks after the onset of the cultures, mineralization is obtained in more than 80% of the marrow cultures. Moreover, morphological studies reveal that during incubation phenotypic changes related to osteogenic differentiation occur at the extracellular matrix as well within cell populations. Well banded collagen is synthesized. Matrix vesicles and needles of hydroxy-apatite crystals are observed via transmission electron microscopy. Osteoblast-like cells are present with membrane-associated alkaline phosphatase activity. the mineralization is specific for cultured bone marrow and is not observed in cultured spleen fragments as is shown via ⁸⁵Sr uptake, calcein uptake and histomorphology. No inducing agent is added to the tissue culture medium except for 10% fetal calf serum, beta-glycerophosphate (10⁻² M) and ascorbic acid. However, the prerequisite for obtaining mineralization is the three-dimensional structure of the marrow in culture. the in vitro organ culture we developed may provide the opportunity to identify which marrow cells have osteogenic potential and to investigate the mechanisms triggering differentiation towards osteogenesis.     

Fetal mouse lung in circumfusion system cultures.

Fetal mouse lungs were cultivated, using the dual-rotary circumfusion system for tissue culture, and their histotypic development was surveyed for 75 days by phase-contrast and electron microscopy. Alveoli, terminal bronchioles and alveolar macrophages were photographed periodically with still and time-lapse phase-contrast microscopy. Their histotypic appearance was confirmed by electron micrographs of the 1- and 2 1/2-month-old specimens. These revealed typical alveoli surrounded by a basal lamina and composed of types I and II pneumocytes containing various lamellar-body forms within the type II cells, the alveolar lumen, and the alveolar macrophages. There was a shift from almost all type II cells in the 1-month-old alveoli to the presence of frequent type I cells as constituents of the alveoli in the 2 1/2-month-old cultures. The terminal bronchioles were tubules consisting of ciliated cells with Clara cells interspersed between them. The ciliated cells contained as many as 30 cilia or basal bodies per section and numerous microvilli. They were attached to each other and to the Clara cells by junctional complexes and accessory desmosomes which were generally in the apical ends of the cells. The Clara cells typically had glycogen granules interspersed between lamellae of the endoplasmic reticulum, contained numerous well dispersed mitochondria, occasional lysosome-like granules and crystalloid bodies which appeared to be tubular. Some Clara cells presented a moderatley dense secretory granule in the center of the whorl of the endoplasmic reticulum.     

Segmental bone replacement via patient‐specific,three‐dimensional printed bioresorbable graft substitutes and their use as templates for the culture of mesenchymal stem cells under mechanical stimulation at various frequencies

The treatment of large segmental bone defects remains a challenge as infection, delayed union, and nonunion are common postoperative complications. A three‐dimensional printed bioresorbable and physiologically load‐sustaining graft substitute was developed to mimic native bone tissue for segmental bone repair. Fabricated from polylactic acid, this graft substitute is novel as it is readily customizable to accommodate the particular size and location of the segmental bone of the patient to be replaced. Inspired by the structure of the native bone tissue, the graft substitute exhibits a gradient in porosity and pore size in the radial direction and exhibit mechanical properties similar to those of the native bone tissue. The graft substitute can serve as a template for tissue constructs via seeding with stem cells. The biocompatibility of such templates was tested under in vitro conditions using a dynamic culture of human mesenchymal stem cells. The effects of the mechanical loading of cell‐seeded templates under in vitro conditions were assessed via subjecting the tissue constructs to 28 days of daily mechanical stimulation. The frequency of loading was found to have a significant effect on the rate of mineralization, as the alkaline phosphatase activity and calcium deposition were determined to be particularly high at the typical walking frequency of 2 Hz, suggesting that mechanical stimulation plays a significant role in facilitating the healing process of bone defects. Utilization of such patient‐specific and biocompatible graft substitutes, coupled with patient’s bone marrow cells seeded and exposed to mechanical stimulation of 2 Hz have the potential of reducing significant volumes of cadaveric tissue required, improving long‐term graft stability and incorporation, and alleviating financial burdens associated with delayed or failed fusions of long bone defects.     

Prolonged culture of HOS 58 human osteosarcoma cells with 1,25-(OH)2-D3, TGF-beta, and dexamethasone reveals physiological regulation of alkaline phosphatase, dissociated osteocalcin gene expression, and protein synthesis and lack of mineralization

Cultured rodent osteoblastic cells reiterate the phenotypic differentiation and maturation of osteoblasts seen in vivo. As previously shown, the human osteosarcoma cell line HOS 58 represents a differentiated stage of osteoblast development. The potential of HOS 58 for still further in vitro differentiation suggests the line can serve as a model of osteoblast maturation. Using this cell line, we have investigated the influence of 1,25-(OH)2-D3 (D3), TGF-beta and Dexamethasone (Dex) on proliferation and on the protein and mRNA levels of alkaline phosphatase (AP), procollagen 1 (Col 1), and osteocalcin (Oc), as well as mineralization during 28 days in culture. AP mRNA and protein were highly expressed throughout the culture period with further increase of protein AP activity at constant gene expression levels. A differentiation inhibiting effect of either TGF-beta or Dex was seen. Col 1 was investigated without the use of ascorbic acid and showed only minor changes during culture time or stimulation. The gene expression for Oc increased continually whereas protein synthesis peaked at confluence and decreased thereafter. TGF-beta and Dex treatments decreased Oc mRNA and protein levels. Stimulation by D3 was maximal at day 7 with a decrease thereafter. HOS 58 cells showed no mineralization capacity when stimulated with different agents, as measured by energy-dispersive X-ray microanalysis. This was not due to absence of Cbfa1 expression. In conclusion, the HOS 58 osteosarcoma cell line represents a differentiated cell line with highly expressed and physiologically regulated AP expression during further differentiation in culture. We observed a dissociation between osteocalcin gene expression and protein secretion which may contribute to the lack of mineralization in this cell line.     

Type II Na+-Pi cotransporters in osteoblast mineral formation: regulation by inorganic phosphate.

During calcification of bone, large amounts of phosphate (P(i)) must be transported from the circulation to the osteoid. Likely candidates for osteoblast P(i) transport are the type II sodium-phosphate cotransporters NaPi-IIa and NaPi-IIb that facilitate transcellular P(i) flux in kidney and intestine, respectively. We have therefore determined the 'cotransporters' expression in osteoblast-like cells. We have also studied the 'cotransporters' regulation by P(i) and during mineralization in vitro. Phosphate uptake and cotransporter protein expression was investigated at early, late and mineralizing culture stages of mouse (MC3T3-E1) and rat (UMR-106) osteoblast-like cells. Both NaPi-IIa and NaPi-IIb were expressed by both osteoblast-like cell lines. NaPi-IIa was upregulated in both cell lines one week after confluency. After 7 days in 3mM P(i) NaPi-IIa was strongly upregulated in both cell lines. NaPi-IIb expression was unaffected by both culture stage and P(i) supplementation. The expression of both cotransporters was unaffected by P(i) deprivation. In vitro mineralization at 1.5mM P(i) was preceded by a three-fold increase in osteoblast sodium-dependent P(i) uptake and a corresponding upregulation of both NaPi-IIa and NaPi-IIb. Their expression thus seem regulated by phosphate in a manner consistent with their playing a role in transcellular P(i) flux during mineralization.     

Fetal mouse lung in circumfusion system cultures

Summary Fetal mouse lungs were cultivated, using the dual-rotary circumfusion system for tissue culture, and their histotypic development was surveyed for 75 days by phase-contrast and electron microscopy. Alveoli, terminal bronchioles and alveolar macrophages were photographed periodically with still and time-lapse phase-contrast microscopy. Their histotypic appearance was confirmed by electron micrographs of the 1- and 2 1/2-month-old specimens. These revealed typical alveoli surrounded by a basal lamina and composed of types I and II pneumocytes containing various lamellar-body forms within the type II cells, the alveolar lumen, and the alveolar macrophages. There was a shift from almost all type II cells in the 1-month-old alveoli to the presence of frequent type I cells as constituents of the alveoli in the 2 1/2-month-old cultures. The terminal bronchioles were tubules consisting of ciliated cells with Clara cells interspersed between them. The ciliated cells contained as many as 30 cilia or basal bodies per section and numerous microvilli. They were attached to each other and to the Clara cells by junctional complexes and accessory desmosomes which were generally in the apical ends of the cells. The Clara cells typically had glycogen granules interspersed between lamellae of the endoplasmic reticulum, contained numerous well dispersed mitochondria, occasional lysosome-like granules and crystalloid bodies which appeared to be tubular. Some Clara cells presented a moderately dense secretory granule in the center of the whorl of the endoplasmic reticulum. This work supported by Grant HL19684 from the National Heart and Lung Institute, National Institutes of Health. Pregnant Strong A mice were kindly supplied by Dr. Henry Browning of the Department of Anatomy.     

Effects of DMEM and RPMI 1640 on the biological behavior of dog periosteum-derived cells

Periosteum-derived cells (PDCs) are being extensively studied as potential tissue engineering seed cells and have demonstrated tremendous promise to date. There is convincing evidence that culture medium could modulate the biological behavior of cultured cells. In this study, we investigate the effects of DMEM (low glucose) and RPMI 1640 on cell growth and cell differentiation of PDCs in vitro. PDCs isolated from Beagle dogs were maintained in DMEM and RPMI 1640, respectively. Then, the cell migration rate of periosteum tissues was analyzed. PDCs of the third passage were harvested for the study of proliferation and osteogenic activity. Proliferation was detected by MTT assay. Alkaline phosphatase activity and mineralized nodules were measured to investigate osteogenic differentiation. Our data demonstrated that DMEM induced alkaline phosphatase activity and strongly stimulated matrix mineralization in cell culture, while similar cell migration rates and proliferation behaviors were observed in the two culture conditions. Interestingly, the osteogenic differentiation of PDCs could be enhanced in DMEM compared with that in RPMI 1640. Thus, it can be ascertained that DMEM may serve as a suitable culture condition allowing osteogenic differentiation of dog PDCs.     

Induction of NK cell activity against fresh human leukemia in culture with interleukin 2

Natural killer (NK) cells have been implicated in defense against malignancies, especially leukemia. Because patients with leukemia and preleukemic disorders manifest low NK activity, it is possible that NK cell impairment may contribute to leukemogenesis. In view of this possibility, it was important to characterize the NK cell defect of leukemic patients and to design new approaches for its correction. Analysis of the mechanism of NK cell defect demonstrated that NK cells of leukemic patients were impaired in their tumor-binding and lytic activity and did not display ability to recycle or to produce cytotoxic factor. However, deficient NK activity could be corrected by culture of peripheral blood effector cells with IL 2. IL 2-activated NK cells manifested restoration of all measured parameters of the cytotoxic mechanism, as exemplified by normalized tumor-binding and lytic activity, as well as the rate of lysis and ability to recycle. Importantly, such in vitro stimulated cytotoxic cells displayed reactivity against fresh leukemic cells of autologous as well as allogeneic origin. Another interesting observation from these studies was that the NK activity was also induced in the leukemic bone marrow, a tissue with a very low frequency of cytotoxic NK cells. It is important to note that cultured NK cells did not represent a stationary cell population, but proliferated in vitro quite actively (doubling time 3 to 6 days) for at least 5 wk. Characterization of the in vitro generated cytotoxic cells indicated that these cells displayed large granular lymphocyte morphology and CD16 and Leu-19 cell surface phenotype. Our data demonstrate that the NK cell defect of leukemic patients is not a permanent phenomenon, but can be reversed in culture with IL 2, and that fully cytotoxic NK cells can be maintained and expanded in vitro. Thus, it is reasonable to suggest that adoptive transfer of autologous NK cells to the patients may represent a promising new therapy for treatment of leukemia.     

Fucose in alpha(1-6)-linkage regulates proliferation and histogenesis in reaggregated retinal spheroids of the chick embryo.

We have used the lectin from Aleuria aurantia (AAL) which is highly specific for alpha(1-6)-linked fucose, to examine its effect on chicken retinogenesis in a reaggregation culture system. When dispersed cells of the embryonic chick retina are reaggregated to form histotypic retinospheroids, AAL elicits strong inhibition of spheroid growth. The action of AAL is specific, since its effect is dose-dependent, saturable, and inhibited by an excess of fucose. Fucosidase treatment entirely abolishes reaggregation. In contrast, Anguilla anguilla agglutinin (AAA) binding to fucose in alpha(1-2)-linkage does not show any effects. Incubation with CAB4-a specific monoclonal antibody for fucose in alpha(1-6)-linkage-reduces spheroid size and shape. AAL does not much affect primary aggregation, but rather subsequent processes of cell proliferation and histogenesis. In particular, AAL inhibits uptake of bromo-desoxyuridine (BrdU), most efficiently so during days in vitro 2 (div2) and div3. As a consequence, the histological differentiation is entirely disturbed, as evidenced by vimentin immunostaining; particularly, rosettes are not forming and the radial glia scaffold is disorganized. We conclude that glycoproteins exhibiting fucose in alpha(1-6)-linkage may play major roles in early processes of retinal tissue formation.     

Long-term growth of lymphokine-activated killer (LAK) cells: role of anti-CD3, beta-IL 1, interferon-gamma and -beta

Peripheral blood lymphocytes (PBL) cultured in interleukin 2 (IL 2)-containing medium in conventional tissue culture develop the ability to lyse fresh tumor cells; such cells are referred to as lymphokine-activated killer (LAK) cells. LAK activity peaks by day 5 of culture and declines rapidly thereafter. We studied culture conditions and signals that allow for long-term culture and expansion of cells with LAK activity. By culturing cells at relatively low densities and regularly replenishing medium and recombinant IL 2 (r-IL 2), LAK function is significantly higher as compared with short-term cultures, and remains present for at least 21 days while cell numbers undergo an average 100-fold expansion. By activating these cultures with anti-CD3 (OKT3) monoclonal antibody and r-IL 2, an approximately 1000-fold expansion in the cell number is obtained with maintenance of comparable levels of LAK activity. The exogenous addition of beta interleukin 1 (beta-IL 1), interferon-beta (IFN-beta) or interferon-gamma (IFN-gamma) can augment the lytic activity of cell populations expanded by anti-CD3 plus r-IL 2. These approaches may enable the in vitro generation from individual donors of much greater numbers of LAK cells for adoptive immunotherapy than can now be obtained with the 3 to 5 day in vitro culture systems.     

Cell disorganization and malformation in neural retina caused by antibodies to R-cognin: ultrastructural study

Retina tissue from 6-day chick embryos was organ-cultured for 3 days in the presence of antibodies to R-cognin, a surface antigen of retina cells. The antibodies which are known to bind to this antigen caused a striking malformation: interruption of the outer limiting membrane and extensive cell disorganization resulting in exteriorization of many cells and forming of chaotic masses on the surface of the tissue. Controls did not show these effects. These results further confirm that R-cognin is involved in the mechanism of histotypic contacts and recognition of retina cells, and that it plays an essential role in cell organization and histogenesis in the retina.