Microwell Culture System Research Articles

Spheroids as a model for endometriotic lesions.

The development and progression of endometriotic lesions are poorly understood, but immune cell dysfunction and inflammation are closely associated with the pathophysiology of endometriosis. There is a need for 3D in vitro models to permit the study of interactions between cell types and the microenvironment. To address this, we developed endometriotic spheroids (ES) to explore the role of epithelial-stromal interactions and model peritoneal invasion associated with lesion development. Using a nonadherent microwell culture system, spheroids were generated with immortalized endometriotic epithelial cells (12Z) combined with endometriotic stromal (iEc-ESC) or uterine stromal (iHUF) cell lines. Transcriptomic analysis found 4,522 differentially expressed genes in ES compared with spheroids containing uterine stromal cells. The top increased gene sets were inflammation-related pathways, and an overlap with baboon endometriotic lesions was highly significant. Finally, to mimic invasion of endometrial tissue into the peritoneum, a model was developed with human peritoneal mesothelial cells in an extracellular matrix. Invasion was increased in the presence of estradiol or pro-inflammatory macrophages and suppressed by a progestin. Taken together, our results strongly support the concept that ES are an appropriate model for dissecting mechanisms that contribute to endometriotic lesion development.

Living on the edge: how stromal cells restrict liver cancer invasion

The global incidence of primary liver cancer is increasing and currently ranks amongst the top-five causes of premature death from cancer annually. Like in many solid tumours, liver cancer cells are closely associated with stromal cells, known as non-parenchymal cells (NPCs), in the liver, including fibroblasts and endothelial cells, which normally support liver function and structure. Although single-cell RNA sequencing has begun to unpick the interactions between tumour cells and this non-epithelial microenvironment, these -omic approaches cannot mechanistically resolve how the non-parenchymal stroma interacts with tumour cells, nor can they resolve whether a fibroblast exerts differential outcomes on mutant versus healthy epithelial cells.In this issue of Disease Models & Mechanisms, Liqin Zhu and colleagues describe a novel in vitro microwell culture system to co-culture NPCs with either healthy hepatocytes or tumour cells of intrahepatic cholangiocarcinoma (iCCA), an aggressive type of primary liver cancer. When the authors co-cultured NPCs with primary hepatocytes, the NPCs supported hepatocyte spheroid growth, maintaining the phenotype of the healthy hepatocytes in culture. Interestingly, when cultured with mutant iCCA cells, NPCs supressed the growth of cancer spheroids, suggesting a divergent role of NPCs in tumour growth.To understand the role of these stromal cells in tumour growth further, the authors then co-cultured healthy hepatocytes and tumour cells in their microwell system and found that tumour cells invade and kill healthy hepatocytes. The addition of NPCs to this system significantly limited this process, and in vitro cancer cells were constrained, no longer killing healthy hepatocytes. Following on from this, the authors used an orthotopic transplantation mouse model of liver cancer and liver tumour samples from patients to show that activated myofibroblasts localised at the interface between tumour and normal tissue. Together, the data suggest that, in liver cancer, NPCs, in particular myofibroblasts, form a physical barrier that limits the ability of cancer cells to invade and damage normal tissue.Targeting the tumour microenvironment to limit disease progression is an attractive therapeutic prospect that could be agnostic to the underlying genetic and molecular heterogeneity seen in cancer. However, as NPCs have previously been shown to provide pro-proliferative signals in cancer and modify the intratumoral immune landscape, more research is required to understand the nuanced NPC-epithelial cell relationship in cancer. We should carefully consider the diverse and potentially conflicting roles of NPCs in tumour progression, and advanced in vitro systems, such as those developed by Zhu and colleagues, will help us decipher this.

Generation of Cortical Brain Organoid with Vascularization by Assembling with Vascular Spheroid.

Background and ObjectivesBrain organoids have the potential to improve our understanding of brain development and neurological disease. Despite the importance of brain organoids, the effect of vascularization on brain organoids is largely unknown. The objective of this study is to develop vascularized organoids by assembling vascular spheroids with cerebral organoids.Methods and ResultsIn this study, vascularized spheroids were generated from non-adherent microwell culture system of human umbilical vein endothelial cells, human dermal fibroblasts and human umbilical cord blood derived mesenchymal stem cells. These vascular spheroids were used for fusion with iPSCs induced cerebral organoids. Immunostaining studies of vascularized organoids demonstrated well organized vascular structures and reduced apoptosis. We showed that the vascularization in cerebral organoids up-regulated the Wnt/β-catenin signaling.ConclusionsWe developed vascularized cerebral organoids through assembly of brain organoids with vascular spheroids. This method could not only provide a model to study human cortical development but also represent an opportunity to explore neurological disease.

402.1: Towards a GMP-Compliant Protocol for the Differentiation of Human Pluripotent Stem Cells to Beta-like Cells for the Treatment of Type 1 Diabetes

Background: β-cell replacement therapy by allogeneic pancreas or islet transplantation is a promising approach for patients with type 1 diabetes. However, there is a scarcity of organ donors. The generation of insulin-producing pancreatic β-cells from human pluripotent stem cells (hPSC) in vitro would provide an unlimited cell source for drug discovery and cell replacement therapy for diabetes. Here we aim to generate β-cells under GMP (Good Manufacturing Practice)-compliant conditions that can be used for clinical application. Methods: We applied a modified seven-stage (30-day) differentiation protocol to generate hPSC-derived insulin-producing β-like cells in a 3D microwell culture system. Next, in order to scale up the production of hPSC-derived β-cells, we adapted the protocol to spinner flask culture. Differentiation was assessed by qPCR, immunocytochemistry and flowcytometry for stage-specific marker expression. β-cell function was evaluated in vitro by GSIS (Glucose-Stimulated Insulin Secretion), and in vivo by intraperitoneal glucose tolerance tests after transplantation of stage-7 cell clusters in immunodeficient mice and human C-peptide secretion was determined by ELISA. Results: In the microwell set-up, cells acquired a definitive endoderm phenotype at stage 1 (day 3), characterized by expression of SOX17 (87.2% ± 4.6; n=5), FOXA2 (82.8% ± 4.6; n=3), co-expression of c-KIT and CXCR4 (93.3% ± 5.6; n=4), and silencing of pluripotency marker OCT4 (2.8% ± 6; n=5). At the end of stage 4 (day 12), pancreatic progenitor cells were identified by the co-expression of PDX1/NKX6.1 (46.7% ± 3.5; n=3). Finally, at the end of stage 7 (day 30), C-peptide-positive β-like cells (49.5% ± 10.5; n=3) and glucagon-positive α-like cells (18.4%± 6.1; n=3) were present. The stimulation index upon exposure to glucose was 4.4 ± 3.1(n=5). Following transplantation of day-30 clusters into mice, stimulated human C-peptide levels reached 46 pmol/L (n=21), 70.5 pmol/L (n=19) and 327.4 pmol/L (n=11) at day 14, 28 and 60 respectively, indicating further maturation of the cells in vivo after transplantation. Next, we scaled up the cell culture in disposable spinner flasks, and obtained comparable differentiation efficiency. Conclusion: We can generate functional hPSCs-derived β-like cells in GMP-compliant conditions in vitro. We will apply these protocols on a clinical-grade PSC line that is currently in expansion (banking) in the GMP facility of our institute. hPSCs-derived β-like cells are a promising future alternative to donor islets for the treatment of type 1 diabetes.

P–269 Effect of well-of-the-well culture as in vitro maturation system for human oocytes

Abstract Study question Can the Well-of-the-Well system (WOW), applied on denuded oocytes, improve germinal vesicle breakdown (GVBD) and maturation rate? Summary answer In vitro maturation (IVM) of denuded germinal vesicle (GV) oocyte using WOW culture system increases nuclear maturation competence when compared with droplet conventional culture What is known already Further research remains necessary to address the mechanism of oocyte maturation in order to refine culture conditions and improve the implantation rate of in vitro matured oocytes. Several studies on bovine oocytes have shown that oocyte-secreted factors (an uncharacterized mix of growth factors secreted by the oocyte) enhance oocyte developmental competence during in vitro maturation. These oocyte-secreted factors may accumulate at the bottom of the micro-well, as suggested for the WOW culture system. Previous reports suggested that diffusible factors secreted by individual oocytes probably accumulated in a micro-well WOW dish, may provide a suitable microenvironment for their in vitro maturation. Study design, size, duration A total of 879 GV collected between 2017 and 2019 were included in this study. They were randomly allocated into two experimental groups: (1) single-cultured oocytes (SC) that were cultured individually in micro-droplets, and (2) group-cultured oocytes (WOW) that were cultured in a microwell culture system using the WOW dish (culture dish for time lapse incubator). The nuclear maturation was assessed after 24 hours and 48 hours of IVM Participants/materials, setting, methods GV oocytes were obtained from 609 patients undergoing controlled ovarian stimulation cycles. Oocytes from the experimental group (1) were placed individually in conventional 25μl micro-droplets in a 35 mm dish. Oocytes from the experimental group (2) were placed in 80 μl droplet individually in each of 9 microwells of WOW dish. All GV oocytes were matured in a single step embryo culture medium, supplemented with human menopausal gonadotropin and synthetic serum substitute. Main results and the role of chance Mature oocyte (MII) was considered when we observed rupture of the GV and the presence of a first polar body in the perivitelline space during the first 24 or 48 hours of culture under inverted optical microscope. GVBD noted significant differences (p-valor = 0.000) between the study groups after culturing of 24 hours [GVBD: SC group; 70% (318/455) vs. WOW group; 83% (352/424)] and 48 hours [GVBD: SC group; 77% (319/416) vs. WOW group; 94% (398/424)]. The maturation rates (MR) showed significant differences (p-valor = 0.000) between the study groups after culturing of 24 hours [MR: SC group; 51% (233/455) vs. WOW group; 80% (338/424)] and 48 hours [MR: SC group; 71% (295/416) vs. WOW group; 91% (387/424)]. Limitations, reasons for caution There is no data on cleavage and blastocyst rates. There are no previous reports comparing the maturation rates in denuded human oocytes single-cultured in individually droplet or group-cultured in WOW dish. Wider implications of the findings: Our results must be taken into account in order to improve the culture conditions for the optimization of the in vitro maturation technique in human oocytes from stimulated cycles. We now provide evidence that group-cultured oocytes in WOW dish increase GVBD and maturation rates. Trial registration number Not applicable

3D Microwell Platform for Cardiomyocyte Differentiation of Human Pluripotent Stem Cells.

The generation of cardiomyocytes (CMs) from human pluripotent stem cells (hPSCs) represents a valuable tool for a myriad of in vitro applications, including drug screening, disease modeling and regenerative medicine. However, the success of these applications is dependent on the establishment of reliable, efficient, simple, and cost-effective differentiation methods. In this chapter, we describe an efficient and robust 3D platform for the generation of hPSC-CMs based on the use of a microwell culture system, which can be applied in any laboratory environment. Additionally, we will also describe protocols for the structural and functional characterization of the obtained CMs for further quality control upon differentiation.

A high-throughput microfluidic method for fabricating aligned collagen fibrils to study Keratocyte behavior.

In vivo, keratocytes are surrounded by aligned type I collagen fibrils that are organized into lamellae. A growing body of literature suggests that the unique topography of the corneal stroma is an important regulator of keratocyte behavior. In this study we describe a microfluidic method to deposit aligned fibrils of type I collagen onto glass coverslips. This high-throughput method allowed for the simultaneous coating of up to eight substrates with aligned collagen fibrils. When these substrates were integrated into a PDMS microwell culture system they provided a platform for high-resolution imaging of keratocyte behavior. Through the use of wide-field fluorescence and differential interference contrast microscopy, we observed that the density of collagen fibrils deposited was dependent upon both the perfusion shear rate of collagen and the time of perfusion. In contrast, a similar degree of fibril alignment was observed over a range of shear rates. When primary normal rabbit keratocytes (NRK) were seeded on substrates with a high density of aligned collagen fibrils and cultured in the presence of platelet derived growth factor (PDGF) the keratocytes displayed an elongated cell body that was co-aligned with the underlying collagen fibrils. In contrast, when NRK were cultured on substrates with a low density of aligned collagen fibrils, the cells showed no preferential orientation. These results suggest that this simple and inexpensive method can provide a general platform to study how simultaneous exposure to topographical and soluble cues influence cell behavior.

Generation of Porcine Testicular Organoids with Testis Specific Architecture using Microwell Culture.

Organoids are three dimensional structures composed of multiple cell types that are capable of recapitulating tissue architecture and functions of organs in vivo. Formation of organoids has opened up different avenues of basic and translational research. In recent years, testicular organoids have garnered interest in the field of male reproductive biology. Testicular organoids allow for the study of cell-cell interactions, tissue development, and the germ cell niche microenvironment and facilitate high throughput drug and toxicity screening. A method is needed to reliably and reproducibly generate testicular organoids with testis specific tissue architecture. The microwell culture system contains a dense array of pyramid-shaped microwells. Testicular cells derived from pre-pubertal testes are centrifuged into these microwells and cultured to generate testicular organoids with testis-specific tissue architecture and cell associations. Thousands of homogeneous organoids can be generated via this process. The protocol reported here will be of broad interest to researchers studying male reproduction.

Recapitulation of hepatitis B virus-host interactions in liver organoids from human induced pluripotent stem cells.

Therapies against hepatitis B virus (HBV) have improved in recent decades; however, the development of individualized treatments has been limited by the lack of individualized infection models. In this study, we used human induced pluripotent stem cell (hiPSC) to generate a functional liver organoid (LO) that inherited the genetic background of the donor, and evaluated its application in modeling HBV infection and exploring virus–host interactions. To establish a functional hiPSC-LO, we cultured hiPSC-derived endodermal, mesenchymal, and endothelial cells with a chemically defined medium in a three-dimensional microwell culture system. Based on cell-cell interactions, these cells could organize themselves and gradually differentiate into a functional organoid, which exhibited stronger hepatic functions than hiPSC derived hepatic like cell (HLC). Moreover, the functional LO demonstrated more susceptibility to HBV infection than hiPSC-HLC, and could maintain HBV propagation and produce infectious virus for a prolonged duration. Furthermore, we found that virus infection could cause hepatic dysfunction of hiPSC-LOs, with down-regulation of hepatic gene expression, induced release of early acute liver failure markers, and altered hepatic ultrastructure. Therefore, our study demonstrated that HBV infection in hiPSC-LOs could recapitulate virus life cycle and virus induced hepatic dysfunction, suggesting that hiPSC-LOs may provide a promising individualized infection model for the development of individualized treatment for hepatitis.

A microwell culture system that allows group culture and is compatible with human single media.

A microwell culture system that facilitates group culture, such as well-of-the-well (WOW), improves embryonic development in an individual culture. We examined the effect of WOW on embryonic development in vitro with commercially available human single culture media. Using four different commercial human single culture media, in vitro development and imprinted gene expression of bovine embryos cultured in WOW were compared to droplet culture (one zygote per drop). To determine the effects of microwell and group culture on embryonic development, different numbers of embryos were cultured in droplet or WOW. Diffusion simulation of accumulating metabolites was conducted using the finite volume method. WOW had a positive effect on bovine embryonic development, regardless of the type of single culture media. Imprinted gene expression was not different between droplet- and WOW-derived blastocysts. The microwell and group cultures in WOW showed a significant positive effect on the rate of total blastocysts and the rate of development to the expanded and hatching blastocyst stages. The assumed cumulative metabolite concentration of WOW with one embryo was 1.47 times higher than that of droplet culture with one embryo. Furthermore, the concentration of WOW with three embryos was 1.54 times higher than that of WOW with one embryo. In using human single culture media, a microwell culture system that allows group culture could be a powerful clinical tool for improving the success of assisted reproductive technologies.

Human liver organoids generated with single donor-derived multiple cells rescue mice from acute liver failure

BackgroundAcute liver failure (ALF) is a life-threatening disease with a high mortality rate. However, there are limited treatments or devices available for ALF therapy. Here, we aimed to develop a new strategy for ALF treatment by transplanting functional liver organoids (LOs) generated from single donor-derived human induced pluripotent stem cell (hiPSC) endoderm, endothelial cells (ECs), and mesenchymal cells (MCs).MethodsFirst, we isolated ECs and MCs from a single donor umbilical cord (UC) through enzyme digestion and characterized the UC-ECs and UC-MCs by flow cytometry. Second, using a nonviral reprogramming method, we generated same donor-derived hiPSCs from the UC-ECs and investigated their hepatic differentiation abilities. Finally, we simultaneously plated EC-hiPSC endoderm, UC-ECs, and UC-MCs in a three-dimensional (3D) microwell culture system, and generated single donor cell-derived differentiated LOs for ALF mouse treatment.ResultsWe obtained ECs and MCs from a single donor UC with high purity, and these cells provided a multicellular microenvironment that promoted LO differentiation. hiPSCs from the same donor were generated from UC-ECs, and the resultant EC-hiPSCs could be differentiated efficiently into pure definitive endoderm and further into hepatic lineages. Simultaneous plating of EC-hiPSC endoderm, UC-ECs, and UC-MCs in the 3D microwell system generated single donor cell-derived LOs (SDC-LOs) that could be differentiated into functional LOs with enhanced hepatic capacity as compared to that of EC-hiPSC-derived hepatic-like cells. When these functional SDC-LOs were transplanted into the renal subcapsules of ALF mice, they rapidly assumed hepatic functions and improved the survival rate of ALF mice.ConclusionThese results demonstrate that functional LOs generated from single donor cells can improve the condition of ALF mice. Functional SDC-LO transplantation provides a promising novel approach for ALF therapy.

Modulation of Wnt/β-catenin signaling in human embryonic stem cells using a 3-D microwell array

Intercellular interactions in the cell microenvironment play a critical role in determining cell fate, but the effects of these interactions on pathways governing human embryonic stem cell (hESC) behavior have not been fully elucidated. We and others have previously reported that 3-D culture of hESCs affects cell fates, including self-renewal and differentiation to a variety of lineages. Here we have used a microwell culture system that produces 3-D colonies of uniform size and shape to provide insight into the effect of modulating cell–cell contact on canonical Wnt/β-catenin signaling in hESCs. Canonical Wnt signaling has been implicated in both self-renewal and differentiation of hESCs, and competition for β-catenin between the Wnt pathway and cadherin-mediated cell–cell interactions impacts various developmental processes, including the epithelial–mesenchymal transition. Our results showed that hESCs cultured in 3-D microwells exhibited higher E-cadherin expression than cells on 2-D substrates. The increase in E-cadherin expression in microwells was accompanied by a downregulation of Wnt signaling, as evidenced by the lack of nuclear β-catenin and downregulation of Wnt target genes. Despite this reduction in Wnt signaling in microwell cultures, embryoid bodies (EBs) formed from hESCs cultured in microwells exhibited higher levels of Wnt signaling than EBs from hESCs cultured on 2-D substrates. Furthermore, the Wnt-positive cells within EBs showed upregulation of genes associated with cardiogenesis. These results demonstrate that modulation of intercellular interactions impacts Wnt/β-catenin signaling in hESCs.

138 TIME-LAPSE CINEMATOGRAPHY-COMPATIBLE INJECTION-MOLDED MICROWELL CULTURE SYSTEM FOR TRACKING THE DEVELOPMENT OF INDIVIDUAL BOVINE EMBRYOS

We have developed a polystyrene-based well of-the-well system (WOW) using injection moulding to track individual embryos throughout culture using time-lapse cinematography (TLC). The WOW-cultured bovine embryos following in vitro fertilization (IVF) were compared with conventional droplet (control)-cultured embryos on in vitro and in vivo development. Twenty-five of zygotes were cultured in each culture system containing 125 μL of CR1aa medium supplemented with 5% calf serum for 168 h after IVF. No differences between control- and WOW-cultured embryos were observed during development to the blastocyst stage. Morphological quality, inner cell mass (ICM), and trophectoderm (TE) cell numbers and post-vitrification survival rates were not different between control- and WOW-derived blastocysts; however, incidence of apoptosis in the ICM and TE cells was reduced in WOW culture (P < 0.01). Oxygen consumption in WOW-derived blastocysts was closer to physiological level than that of control-derived blastocysts. Moreover, WOW culture improved embryo viability, as indicated by increased pregnancy rates at days 30 and 60 after embryo transfer (P < 0.05). The TLC monitoring was performed to evaluate the cleavage pattern and the duration of the first cell cycle of embryos from oocytes collected by ovum pick-up; correlations with success of pregnancy were determined. Logistic regression analysis indicated that the cleavage pattern correlated with success of pregnancy (P < 0.05), but cell cycle length did not. Higher pregnancy rates (66.7%) were observed for animals in which transferred blastocysts had undergone normal cleavage, identified by the presence of 2 blastomeres of the same size without fragmentation, than among those with abnormal cleavage (33.3%). These results suggest that our microwell culture system is a powerful tool for producing and selecting healthy embryos and for identifying viability biomarkers. This work was supported by the Research and Developmental Program for New Bio-Industry Initiatives.

Time-Lapse Cinematography-Compatible Polystyrene-Based Microwell Culture System: A Novel Tool for Tracking the Development of Individual Bovine Embryos1

We have developed a polystyrene-based well-of-the-well (WOW) system using injection molding to track individual embryos throughout culture using time-lapse cinematography (TLC). WOW culture of bovine embryos following in vitro fertilization was compared with conventional droplet culture (control). No differences between control- and WOW-cultured embryos were observed during development to the blastocyst stage. Morphological quality and inner cell mass (ICM) and trophectoderm (TE) cell numbers were not different between control- and WOW-derived blastocysts; however, apoptosis in both the ICM and TE cells was reduced in WOW culture (P < 0.01). Oxygen consumption in WOW-derived blastocysts was closer to physiological level than that of control-derived blastocysts. Moreover, WOW culture improved embryo viability, as indicated by increased pregnancy rates at Days 30 and 60 after embryo transfer (P < 0.05). TLC monitoring was performed to evaluate the cleavage pattern and the duration of the first cell cycle of embryos from oocytes collected by ovum pickup; correlations with success of pregnancy were determined. Logistic regression analysis indicated that the cleavage pattern correlated with success of pregnancy (P < 0.05), but cell cycle length did not. Higher pregnancy rates (66.7%) were observed for animals in which transferred blastocysts had undergone normal cleavage, identified by the presence of two blastomeres of the same size without fragmentation, than among those with abnormal cleavage (33.3%). These results suggest that our microwell culture system is a powerful tool for producing and selecting healthy embryos and for identifying viability biomarkers.

Time-lapse cinematography対応型マイクロウェル培養システム: 単一胚の発生追跡ツール

Time-lapse cinematography対応型マイクロウェル培養システム: 単一胚の発生追跡ツール

Rescue of zona-free embryos by partial encapsulation with collagen followed by coculture with endometrial cells

Zona-free oocytes, a frequent occurrence when micromanipulation techniques are employed in IVF laboratories, are usually discarded. The purpose of this study was to see whether these embryos can be rescued by encapsulation with artificial zona pellucida. Zona-free mouse embryos encapsulated with sodium alginate (AZP, Group A), partially encapsulated with collagen micro-wells (Group B), zona-free embryos (Group C), and zona-intact embryos as control (Group D), were cocultured with endometrial cells. Embryo development of these studied groups were compared. Enzymatically isolated mouse endometrial cells were plated in 4-well plates (2 × 105 cells/well) and cultured with F12/199 medium supplemented with 10% FCS before coculture. Pronuclear-staged embryos were harvested from superovulated female CF1 mice (zona intact, control), treated with pronase to remove the zona pellucida (zona-free). Zona-free embryos were immersed in 1.5% sodium alginate solution, then aspirated with a small amount of sodium alginate and expelled into 1.5% calcium chloride to form microgels (AZP). All zona-intact, zona-free, and AZP embryos were cocultured with endometrial cells plated in 4-well plates directly. For the collagen microwells group, collagen type-I were coated at the surface of the monolayer endometrial cells in the 4-well plates and these micro-wells were formed by introducing small air bubbles in the layer of the collagen gel. The size of the wells is slightly larger then that of the embryos in order to encapsulate the embryos. Zona-free embryos were then transferred to each of the micro-well for coculture (collagen micro-wells group). Embryo development were evaluated every day for 5 days. Zona-free embryos tend to have abnormal early embryo development. They either disassociated into fragments or aggregated to form mosaic embryos. Addition of fully or partially encapsulated zona pellucida (AZP and collagen micro-wells, respectively) did enhance embryo development of these zona-free embryos. Most of the AZP appeared to have abnormal blastomere arrangement and blastocoele collapse and its blastocyst formation rate was compromised. However, collagen group exhibited normal embryo development and resulted in a blastocyst formation rate similar to that of the control. Our data suggests that artificial zona pellucida may rescue some zona-free embryos encountered during IVF. Partial encapsulation of collagen appears to increase cell-cell contacts resulting in normal blastomere polarization and allow space for embryo development. Recently, a new method for zona-free cloning has been demonstrated to increase the efficiency of cloning for stem cell study. Zona-free embryos generated from zona-free cloning can be benefited by culturing in our collagen micro-well culture system to improve embryo quality, thus avoiding monozygotic twinning and preventing adhesion of zona-free embryos to the feeder cells before transfer.

Immunoregulation and blocking antibodies induced by interferon beta treatment in MS.

To examine the in vivo immunoregulatory properties of interferon beta-1a (IFN beta-1a) on the T cell responses to myelin basic protein (MBP) and to evaluate the occurrence of the blocking antibodies characterized by the ability to reverse the effects of IFN beta on T cells in MS patients treated with IFN beta. The precursor frequency of T cells recognizing MBP and control antigens was estimated in a microwell culture system. The cytokine profile of T cell lines was measured in ELISA. The binding antibodies were determined in ELISA and Western blot. Cytopathic test and the T cell functional assays were used to determine the blocking effects of the binding antibodies. Treatment with IFN beta resulted in a substantial reduction in the precursor frequency of MBP-reactive T cells in MS patients. The cytokine profile of MBP-reactive T cells that sustained the treatment was altered toward an increased production of interleukin (IL)-10 and decreased production of tumor necrosis factor (TNF)alpha and IFN-gamma. The immunoregulatory properties of IFN beta on T cells could be blocked by the binding antibodies derived from a proportion of patients treated with IFN beta (4 of 64, 6.25%). The blocking antibodies also neutralized anti-viral activity of IFN beta in cytopathic assays, corresponding to previously described neutralizing antibodies. Treatment with IFN beta alters the cytokine profile by enhancing the production of IL-10 and downregulating Th1 cytokines, which may contribute to clinical benefit in MS. The treatment also induces blocking antibodies that impair the immunoregulatory properties of IFN beta in some individuals.

Induction of immunoglobulin secreting cells in the human autologous mixed leukocyte reaction: regulation by helper and suppressor lymphocyte subsets defined with monoclonal antibodies.

The generation of IgM- and IgG-secreting cells in the human autologous MLR has been confirmed by using a microwell culture system and a reverse hemolytic plaque assay. The regulation of this response by T cell subsets was examined by using monoclonal antibodies that have previously allowed the definition of a Leu-3 helper/inducer subpopulation and a Leu-2 suppressor/cytotoxic subpopulation. These subsets were isolated by "panning" with monoclonal antibodies and were then cultured with autologous non-T cells, which serve as both stimulators of the reaction and a B cell source. The induction of antibody-forming cells was found to be dependent upon the Leu-3 subset whereas the Leu-2 subset not only failed to support C cell maturation, it actively suppressed the Leu-3 induced response. The Leu-3 response was found to be partially sensitive to radiation doses of 1000 rad or greater, whereas Leu-2 mediated suppression, even at high doses of cells, was totally ablated by 1500 rad. Thus, the generation of antibody-forming cells in the human autologous MLR is regulated by a balance of Leu-3 cell help and Leu-2 cell suppression. Such a finding may prove to be of relevance to immunoregulation in both health and disease.

Highly sensitive assay for Clostridium perfringens enterotoxin that uses inhibition of plating efficiency of Vero cells grown in culture.

A highly sensitive and reproducible biological assay for Clostridium perfringens enterotoxin is described that uses Vero (African green monkey kidney) cells grown in tissue cultures. Very small doses of the enterotoxin inhibited the plating efficiency of the cells. This inhibition of plating efficiency could be used to detect as little as 0.1 ng (1 ng/ml) of enterotoxin, and a linear dose-response curve was obtained with 0.5 to 5 ng (5 to 50 ng/ml). A nonlinear, but reproducible, curve was obtained with a dose range from 0.1 to 100 ng (1 to 1,000 ng/ml). A new unit of biological activity, called the plating efficiency unit, was defined as that amount of enterotoxin that caused a 25% inhibition of the plating of 200 cells inoculated into 100 microliters of medium in a microwell culture system. One milligram of highly purified enterotoxin contained about 400,000 plating efficiency units. Additional studies demonstrated that the biological and serological activities of the enterotoxin molecule were not equally labile.

Histogenesis of mouse cerebellum in microwell cultures. Cell reaggregation and migration, fiber and synapse formation.

A microwell culture system was developed for analysis of cell movements and interactions during nervous system histogenesis. Cells from trypsinized 7-day-old mouse cerebellum reaggregated within hours into clusters which later developed interconnections consisting of either sheets of migrating cells and cell processes or cables of fiber bundles with cells migrating along their surfaces. Granule cells in several stages of differentiation, basket and/or stellate neurons, some larger neurons, and two types of neuroglial cells were identified in reproducible, nonrandom patterns by scanning and transmission electron microscopy. Axonal and dendritic processes, both with growth cones, and numerous synapses were generated in vitro.