Organoid Culture Techniques Research Articles

Advancements in Organoid Culture Technologies: Current Trends and Innovations.

Organoids have emerged as valuable tools in investigating disease mechanisms, drug efficacy, and personalized medicine due to their capacity to recapitulate crucial aspects of tissue physiology, including cell-cell interactions, heterogeneity, microenvironmental cues, and drug responses. Despite their broad applicability across various research domains, conventional organoid culture methods are plagued by several limitations that hinder research progress. These limitations include the inability to faithfully recreate tissue microenvironments, immune contexts, and vascular systems. Fortunately, ongoing advancements in organoid culture techniques are addressing these shortcomings. In this review, we provide a comprehensive overview of current mainstream organoid culture protocols. By evaluating these protocols, researchers can identify the most suitable experimental methods, thereby optimizing resource allocation and experimental outcomes.

Differential beta-coronavirus infection dynamics in human bronchial epithelial organoids.

The lower respiratory system serves as the target and barrier for beta-coronavirus (beta-CoV) infections. In this study, we explored beta-CoV infection dynamics in human bronchial epithelial (HBE) organoids, focusing on HCoV-OC43, SARS-CoV, MERS-CoV, and SARS-CoV-2. Utilizing advanced organoid culture techniques, we observed robust replication for all beta-CoVs, particularly noting that SARS-CoV-2 reached peak viral RNA levels at 72 h postinfection. Through comprehensive transcriptomic analysis, we identified significant shifts in cell population dynamics, marked by an increase in goblet cells and a concurrent decrease in ciliated cells. Furthermore, our cell tropism analysis unveiled distinct preferences in viral targeting: HCoV-OC43 predominantly infected club cells, while SARS-CoV had a dual tropism for goblet and ciliated cells. In contrast, SARS-CoV-2 primarily infected ciliated cells, and MERS-CoV showed a marked affinity for goblet cells. Host factor analysis revealed the upregulation of genes encoding viral receptors and proteases. Notably, HCoV-OC43 induced the unfolded protein response pathway, which may facilitate viral replication. Our study also reveals a complex interplay between inflammatory pathways and the suppression of interferon responses during beta-CoV infections. These findings provide insights into host-virus interactions and antiviral defense mechanisms, contributing to our understanding of beta-CoV infections in the respiratory tract.

Derivation of pancreatic acinar cell carcinoma cell line HS-1 as a patient-derived tumor organoid.

Acinar cell carcinoma (ACC) of the pancreas is a malignant tumor of the exocrine cell lineage with a poor prognosis. Due to its rare incidence and technical difficulties, few authentic human cell lines are currently available, hampering detailed investigations of ACC. Therefore, we applied the organoid culture technique to various types of specimens, such as bile, biopsy, and resected tumor, obtained from a single ACC patient. Despite the initial propagation, none of these organoids achieved long-term proliferation or tolerated cryopreservation, confirming the challenging nature of establishing ACC cell lines. Nevertheless, the biopsy-derived early passage organoid developed subcutaneous tumors in immunodeficient mice. The xenograft tumor histologically resembled the original tumor and gave rise to infinitely propagating organoids with solid features and high levels of trypsin secretion. Moreover, the organoid stained positive for carboxylic ester hydrolase, a specific ACC marker, but negative for the duct cell marker CD133 and the endocrine lineage marker synaptophysin. Hence, we concluded the derivation of a novel ACC cell line of the pure exocrine lineage, designated HS-1. Genomic analysis revealed extensive copy number alterations and mutations in EP400 in the original tumor, which were enriched in primary organoids. HS-1 displayed homozygous deletion of CDKN2A, which might underlie xenograft formation from organoids. Although resistant to standard cytotoxic agents, the cell line was highly sensitive to the proteasome inhibitor bortezomib, as revealed by an in vitro drug screen and in vivo validation. In summary, we document a novel ACC cell line, which could be useful for ACC studies in the future.

Colon cancer organoids using monoclonal organoids established in four different lesions of one cancer patient reveal tumor heterogeneity and different real-time responsiveness to anti-cancer drugs.

Organoid culture technique has been taking center stage as a next-generation ex-vivo model due to advancement of stem cell research techniques. The importance of the laboratory-based ex vivo model has increasingly been recognized for recapitulating histological, and physioglocal conditions of in vivo microenviorment. Accordingly, the use of this technique has also broadened the understanding of intratumoral heterogeneity which is closely associated with varied drug responses observed in patients. Likewise, studies on heterogeneity within a single tumor tissue have drawn much attention. Here, we isolated 15 single clones from 4 tumor organoid lines from 1 patient at a primary passage from one patient. Each organoid line showed variable alterations in both genotype and phenotype. Furthermore, our methodological approach on drug test employing a high-throughput screening system enabled us to pinpoint the optimal time frame for anti-cancer drugs within a single tumor. We propose that our method can effectively reveal the heterogeneity of time-point in drug response, and the most optimal therapeutic strategies for individual patient.

Tumor organoids: synergistic applications, current challenges, and future prospects in cancer therapy.

Patient‐derived cancer cells (PDCs) and patient‐derived xenografts (PDXs) are often used as tumor models, but have many shortcomings. PDCs not only lack diversity in terms of cell type, spatial organization, and microenvironment but also have adverse effects in stem cell cultures, whereas PDX are expensive with a low transplantation success rate and require a long culture time. In recent years, advances in three‐dimensional (3D) organoid culture technology have led to the development of novel physiological systems that model the tissues of origin more precisely than traditional culture methods. Patient‐derived cancer organoids bridge the conventional gaps in PDC and PDX models and closely reflect the pathophysiological features of natural tumorigenesis and metastasis, and have led to new patient‐specific drug screening techniques, development of individualized treatment regimens, and discovery of prognostic biomarkers and mechanisms of resistance. Synergistic combinations of cancer organoids with other technologies, for example, organ‐on‐a‐chip, 3D bio‐printing, and CRISPR‐Cas9‐mediated homology‐independent organoid transgenesis, and with treatments, such as immunotherapy, have been useful in overcoming their limitations and led to the development of more suitable model systems that recapitulate the complex stroma of cancer, inter‐organ and intra‐organ communications, and potentially multiorgan metastasis. In this review, we discuss various methods for the creation of organ‐specific cancer organoids and summarize organ‐specific advances and applications, synergistic technologies, and treatments as well as current limitations and future prospects for cancer organoids. Further advances will bring this novel 3D organoid culture technique closer to clinical practice in the future.

Advance in Human Epithelial-Derived Organoids Research.

Organoids have complex three-dimensional structures that exhibit functionalities and feature architectures similar to those of in vivo organs and are developed from adult stem cells, embryonic stem cells, and pluripotent stem cells through a self-organization process. Organoids derived from adult epithelial stem cells are the most mature and extensive. In recent years, using organoid culture techniques, researchers have established various adult human tissue-derived epithelial organoids, including intestinal, colon, lung, liver, stomach, breast, and oral mucosal organoids, all of which exhibit strong research and application prospects. Studies have shown that epithelial organoids are mainly applied in drug discovery, personalized drug response testing, disease mechanism research, and regenerative medicine. In this review, we mainly discuss current organoid culture systems and potential applications of this technique with human epithelial tissue.

Fine-Needle Aspiration-Based Patient-Derived Cancer Organoids.

SummaryPatient-derived cancer organoids hold great potential to accurately model and predict therapeutic responses. Efficient organoid isolation methods that minimize post-collection manipulation of tissues would improve adaptability, accuracy, and applicability to both experimental and real-time clinical settings. Here we present a simple and minimally invasive fine-needle aspiration (FNA)-based organoid culture technique using a variety of tumor types including gastrointestinal, thyroid, melanoma, and kidney. This method isolates organoids directly from patients at the bedside or from resected tissues, requiring minimal tissue processing while preserving the histologic growth patterns and infiltrating immune cells. Finally, we illustrate diverse downstream applications of this technique including in vitro high-throughput chemotherapeutic screens, in situ immune cell characterization, and in vivo patient-derived xenografts. Thus, routine clinical FNA-based collection techniques represent an unappreciated substantial source of material that can be exploited to generate tumor organoids from a variety of tumor types for both discovery and clinical applications.

Sample preparation strategies for high-throughput mass spectrometry imaging of primary tumor organoids.

Patient-derived 3D organoids show great promise for understanding patient heterogeneity and chemotherapy response in human-derived tissue. The combination of organoid culture techniques with mass spectrometry imaging provides a label-free methodology for characterizing drug penetration, patient-specific response, and drug biotransformation. However, current methods used to grow tumor organoids employ extracellular matrices that can produce small molecule background signal during mass spectrometry imaging analysis. Here, we develop a method to isolate 3D human tumor organoids out of a Matrigel extracellular matrix into gelatin mass spectrometry compatible microarrays for high-throughput mass spectrometry imaging analysis. The alignment of multiple organoids in the same z-axis is essential for sectioning organoids together and for maintaining reproducible sample preparation on a single glass slide for up to hundreds of organoids. This method successfully removes organoids from extracellular matrix interference and provides an organized array for high-throughput imaging analysis to easily identify organoids by eye for area selection and further analysis. With this method, mass spectrometry imaging can be readily applied to organoid systems for preclinical drug development and personalized medicine research initiatives.

Stem cell-based therapy for inflammatory bowel disease.

Inflammatory bowel disease (IBD) is an idiopathic, multi-etiological disease characterized by inflammation and mucosal destruction of the gastrointestinal tract. Despite the remarkable advance in immunomodulating therapies, there still remains a certain population of patients who are refractory to conventional as well as biologic therapies and fail to achieve mucosal healing. To improve the prognosis of those patients, at least 2 types of stem cells have been tested for their potential therapeutic use. Transplantation of hematopoietic stem cells or mesenchymal stem cells have been tested in several clinical studies, but their beneficial effect still remains controversial. In this review, we would like to overview the recent clinical challenges of stem cell-based therapies in IBD and also introduce our new therapeutic plan of intestinal stem cell transplantation for IBD, based on our ex vivo intestinal organoid culture technique.

Organoid-based ex vivo reconstitution of Kras-driven pancreatic ductal carcinogenesis.

The organoid culture technique has been recently applied to modeling carcinogenesis in several organs. To further explore its potential and gain novel insights into tumorigenesis, we here investigated whether pancreatic ductal adenocarcinoma (PDA) could be generated as subcutaneous tumors in immunocompromised nude mice, by genetic engineering of normal organoids. As expected, acute induction of KrasG12Din vitro occasionally led to development of tiny nodules compatible with early lesions known as pancreatic intraepithelial neoplasia (PanIN). KrasG12D-expressing cells were enriched after inoculation in the subcutis, yet proved rather declined during culture, suggesting that its advantage might depend on surrounding environments. Depletion of growth factors or concurrent Trp53 deletion resulted in its robust enrichment, invariably leading to development of PanIN or large high-grade adenocarcinoma, respectively, consistent with in vivo mouse studies for the same genotype. Progression from PanIN was also recapitulated by subsequent knockdown of common tumor suppressors, whereas the impact of Tgfbr2 deletion was only partially recapitulated, illustrating genotype-dependent requirement of the pancreatic niche for tumorigenesis. Intriguingly, analysis of tumor-derived organoids revealed that KrasG12D-expressing cells with spontaneous deletion of wild-type Kras were positively selected and exhibited an aging-related mutation signature in nude mice, mirroring the pathogenesis of human PDA, and that the sphere-forming potential and orthotopic tumorigenicity in syngenic mice were significantly augmented. These observations highlighted the relevance of the subcutis of nude mice in promoting PDA development despite its ectopic nature. Taken together, pancreatic carcinogenesis could be considerably recapitulated with organoids, which would probably serve as a novel disease model.

Organoids of liver diseases: From bench to bedside.

Understanding the occurrence, development, and treatment of liver diseases is the main goal of hepatopathology research. Liver diseases are not only diverse but also highly heterogeneous among individuals. At present, research on liver diseases is conducted mainly through cell culture, animal models, pathological specimens, etc. However, these methods cannot fully reveal the pathogenic mechanism and therapeutic characteristics of individualized liver diseases. Recent advances in three-dimensional cell culture technology (organoid culture techniques) include pluripotent stem cells and adult stem cells that are cultured in vitro to form self-organizing properties, making it possible to achieve individualized liver disease research. This review provides a comprehensive overview of the development of liver organoids, the existing and potential applications of liver regenerative medicine, the pathogenesis of liver disease heterogeneity, and drug screening.

Shortcuts to intestinal carcinogenesis by genetic engineering in organoids.

Inactivation of the Adenomatous polyposis coli (APC) gene is an initiating and the most relevant event in most sporadic cases of colorectal cancer, providing a rationale for using Apc‐mutant mice as the disease model. Whereas carcinogenesis has been observed only at the organism level, the recent development of the organoid culture technique has enabled long‐term propagation of intestinal stem cells in a physiological setting, raising the possibility that organoids could serve as an alternative platform for modeling colon carcinogenesis. Indeed, it is demonstrated in the present study that lentivirus‐based RNAi‐mediated knockdown of Apc in intestinal organoids gave rise to subcutaneous tumors upon inoculation in immunodeficient mice. Reconstitution of common genetic aberrations in organoids resulted in development of various lesions, ranging from aberrant crypt foci to full‐blown cancer, recapitulating multi‐step colorectal tumorigenesis. Due to its simplicity and utility, similar organoid‐based approaches have been applied to both murine and human cells in many investigations, to gain mechanistic insight into tumorigenesis, to validate putative tumor suppressor genes or oncogenes, and to establish preclinical models for drug discovery. In this review article, we provide a multifaceted overview of these types of approaches that will likely accelerate and advance research on colon cancer.

Abstract B051: PDX-derived and patient-derived prostate cancer organoids as a clinically relevant platform for translational research

Abstract Metastatic castration-resistant prostate cancer (CRPC) is a genetically heterogeneous disease. Effective translational research demands a diverse and representative set of preclinical models. A major obstacle in the field is the limited capacity to culture prostate cancer-derived cells in vitro. Few cell lines exist. Those that are available do not well represent the genetic diversity of the disease, nor do they accurately predict in vivo response. Recently, organoid culture techniques developed in the Sawyers and Clevers laboratories have increased our ability to grow metastatic tumor-derived prostate cancer cells in vitro. This represents a great step forward, but is restricted by limited availability of tissue and a success rate of <20% when establishing new lines. In the field of prostate cancer, patient-derived xenograft (PDX) lines are a model that broadly represents the clinical and genetic diversity of the disease. Like primary prostate tumors, PDXs have not been readily adaptable to in vitro culture (including the standard organoid method), making them poorly suited for mechanistic studies or high-throughput drug screens. Therefore, there is great interest in adapting the prostate cancer PDX model to an in vitro culture system. The LuCaP series of PDXs is a well-characterized set of ~40 diverse prostate cancer specimens. Using LuCaP PDXs, we systematically tested several modifications to the organoid culture method. This includes the addition of factors to modify metabolism, growth, and differentiation; removal of standard media components; and combinations thereof. We show that p38 is required for growth and survival of almost all the LuCaP organoids. Removal of the p38 inhibitor component from standard prostate organoid media improves initial growth and makes it possible to maintain many PDX-derived organoids over several generations. Additional modifications to individual LuCaP culture conditions improve growth. We successfully cultured 21 of 24 different LuCaPs attempted, for at least one generation, and 10 have been grown long-term. We show that PDX-derived organoids recapitulate the phenotype of the original PDX tumor, including AR-dependent growth. For example, we have shown that homologous recombination deficient LuCaP organoid responses to olaparib reflect the enhanced sensitivity seen for patients in clinical trials. Additionally, our method improves the success rate for culturing samples directly from patient biopsies. We show that 2 of 3 biopsy-derived organoid lines that we have established grow well in our conditions but not in standard published conditions. The third grows equally well in both. Further, our data suggest that p38 is a previously unappreciated target for CRPC treatment. Importantly, our work immediately and significantly increases the number of prostate cancer lines available to the community by making the LuCaP PDXs accessible to in vitro culture, and by improving the success rate for establishing organoids directly from patient samples. Citation Format: Michael L. Beshiri, Caitlin M. Tice, Crystal Tran, Holly M. Nguyen, Adam G. Sowalsky, Supreet Agarwal, Keith H. Jansson, Kerry McGowen, Aian Neil Alilin, JuanJuan Yin, Fatima H. Karzai, William L. Dahut, Eva Corey, Kathleen Kelly. PDX-derived and patient-derived prostate cancer organoids as a clinically relevant platform for translational research [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr B051.

Abstract 4838: Optimization of prostate cancer organoid culture methods to grow PDX and human biopsy tissue

Abstract A major obstacle in the field of prostate cancer research is the limited capacity to culture prostate cancer-derived cells in vitro. There are relatively few established prostate cancer cell lines in use today. Those that are available are amenable to in vitro drug assays and mechanistic studies, but do not well-represent the genetic diversity of the disease, nor do they accurately predict in vivo response. Recently, organoid culture techniques have been developed for prostate cancer by the laboratories of Hans Clevers and Charles Sawyers that have increased our ability to grow metastatic tumor-derived prostate cancer cells in vitro. This represents a great step forward, but we are still restricted by the limited availability of biopsy tissue and the relatively low success rate (~20%) in establishing new organoid lines. The LuCaP series of patient-derived xenografts (PDXs) represents a well-characterized set of prostate cancer specimens that is genetically diverse, reflective of a range of treatment histories, and available to the research community. We have modified the organoid culture technique based upon systematic comparisons and analysis for organoid growth of the LuCaP PDXs. Our modified conditions represent an improvement upon the original growth conditions. We have successfully cultured 21 out of 24 LuCaPs attempted, for at least one generation, and 16 out of 24 are able to grow long-term over several generations. This includes 3 of the newest LuCaPs (167, 170.2, and 189.3) that we have genomically characterized. Comparison of the PDX and various organoid generations showed significant conservation of genomic changes ranging from 75-90% for nonsynonymous point mutations. The modified organoid culture method immediately and significantly increases the number of prostate cancer lines that are available by making the LuCaP PDXs accessible to in vitro culture. In addition to the PDXs, our method has improved the success for culturing metastatic samples grown from biopsies. We show here that 2 out of 3 patient-derived organoid lines that we have established, grow well in the modified conditions but poorly in the original culture conditions, the third grows equally well in both conditions. Citation Format: Mike L. Beshiri, Crystal Tran, Adam G. Sowalsky, Holly M. Nguyen, Supreet Agarwal, Caitlin M. Tice, Eva Corey, Kathy Kelly. Optimization of prostate cancer organoid culture methods to grow PDX and human biopsy tissue [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4838. doi:10.1158/1538-7445.AM2017-4838

Abstract 4378: Gastric cancer organoid culture shows preserved genomic stability in long-term passage

Abstract With recent advances in 3D organoid culture techniques, normal epithelial and tumor cells can be directly cultured from clinical specimens in vitro and expanded long-term with a very high success rate. Thus, this constitutes a good platform for various mechanistic functional studies and clinical applications. A previous karyotyping and gene expression profiling study has indicated that organoid culture of normal epithelial cells can be propagated for long periods of time without genomic alterations. However, data on the genomic stability of tumor organoids in long-term culture are not yet available. We have successfully established organoid cultures derived from either primary tumor or lymph node metastasis from 4 gastric cancer patients. For each sample, we performed long-term culture for at least 6 months. DNA was extracted from blood, as well as the early and late passages of these organoids, and submitted for whole-exome sequencing (WES), achieving a mean coverage approaching 50X. Somatic mutations detected in early versus late passage gastric cancer organoids were compared. Furthermore, we examined the organoids for copy number variations based on the coverage and loss of heterozygosity (LOH) information derived from exome sequencing. We detected between 80 to 228 somatic mutations in the 4 early passage organoids, in which over 84-99% of them were retained during long-term culture. We detected 16, 20, 31 and 98 new mutations in the 4 long-term cultures, respectively. The one case with a substantially large number of new mutations (n=98) appeared to have emerged from a subclone of TP53 wild-type cells in a TP53 mutant tumor. Notably, the C>T mutation was the most dominant mutation spectrum in this case, constituting 48%. Despite the presence of frequent long segment LOH and chromosomal aberrations in early passage organoids, indicating the presence of chromosomal instability, these patterns of aberrations were stably maintained in long-term passage. Overall, with the current organoid culture protocol, tumor genomes are mostly stably maintained with the accumulation of only a small number of new mutations. Our results indicate that this is a reliable and stable in vitro cell culture model for various cancer-related and clinical studies. Citation Format: Sarah Siu Kuen Yue, Helen Hoi Ning Yan, Hoi Cheong Siu, Siu Lun Ho, Wai Yin Tsui, Dessy Chan, Annie Shuk Yee Chan, Bernard Chi Hang Lee, Anthony Kin Wang Chan, Suet Yi Leung. Gastric cancer organoid culture shows preserved genomic stability in long-term passage [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4378. doi:10.1158/1538-7445.AM2017-4378

Intersections of lung progenitor cells, lung disease and lung cancer.

The use of stem cell biology approaches to study adult lung progenitor cells and lung cancer has brought a variety of new techniques to the field of lung biology and has elucidated new pathways that may be therapeutic targets in lung cancer. Recent results have begun to identify the ways in which different cell populations interact to regulate progenitor activity, and this has implications for the interventions that are possible in cancer and in a variety of lung diseases. Today’s better understanding of the mechanisms that regulate lung progenitor cell self-renewal and differentiation, including understanding how multiple epigenetic factors affect lung injury repair, holds the promise for future better treatments for lung cancer and for optimising the response to therapy in lung cancer. Working between platforms in sophisticated organoid culture techniques, genetically engineered mouse models of injury and cancer, and human cell lines and specimens, lung progenitor cell studies can begin with basic biology, progress to translational research and finally lead to the beginnings of clinical trials.

Reconstruction of cartilage tissue using scaffold-free organoid culture technique

We applied the scaffold-free culture method to chondrocytes and attempted to reconstitute articular cartilage grafts. Primary rat costal chondrocytes were immobilized into hollow fibers by centrifugation at a density of 3 x 10(8) cells/cm(3) to induce the formation of cylindrical-shaped multicellular aggregates (organoids) and cultured for one month. The organoids were evaluated by histological and gene expression analyses. Chondrocytes formed cylindrical organoids in hollow fibers (HFs). Histochemical analysis revealed the accumulation of a cartilage extracellular matrix (collagen and proteoglycan) around cells in the lumen of HFs with culture time, forming a low-cellular-density tissue similar to native cartilage by day 28. Furthermore, in contrast to that in traditional monolayer culture, the organoid maintained the gene expression of the cartilage extracellular matrix (type II collagen, aggrecan) for one month of culture. In conclusion, our organoid formation method was effective in producing a cartilage-like tissue. This result suggests that the technique may be applicable to the development of an articular cartilage graft.

Hepatic Differentiation of Embryonic Stem Cells in HF/Organoid Culture

Objective The use of embryonic stem cells (ES cells) has recently received much attention as a novel cell source for various hybrid artificial organs. To use ES cells, it is necessary to be able to produce functional mature cells from ES cells in large quantities. We applied HF/organoid culture, where cultured cells formed cylindrical multicellular aggregates (organoids) in the lumen of hollow fibers, to mouse and cynomolgus monkey ES cells for hepatic differentiation. Materials and Methods ES cells were injected into hollow fibers. The hollow fibers were centrifuged to induce organoid formation and cultured in medium including factors for hepatic differentiation. To determine the characteristics of cells in the bundle, we evaluated gene expression and liver-specific functions. Results ES cells immobilized inside hollow fibers proliferated and formed cylindrical organoids. In mouse ES cell cultures, the expression of mRNAs of hepatocyte-specific genes increased with culture time. Ammonia removal activity detected at 15 days increased with culture time. Albumin secretion activity detected at 12 days increased by 21 days. In cynomolgus monkey ES cell cultures, ES cells showed spontaneous ammonia removal functions. The maximum levels of these functions per unit volume of the hollow fibers were roughly comparable to those of primary hepatocyte-organoids. Conclusions ES cells differentiated into hepatocyte-like cells using the organoid culture technique. The results indicated that the combination of ES cells and an organoid culture technique was useful to obtain mature hepatocytes.

Organoid culture of cannulated rat resistance arteries: effect of serum factors on vasoactivity and remodeling.

We developed an organoid culture technique to study the mechanisms involved in arterial remodeling. Resistance arteries were isolated from rat cremaster muscle and mounted in a pressure myograph at 75 mmHg. Vessels were studied during a 4-day culture period in DMEM with either 2% albumin, 10% heat-inactivated FCS (HI-FCS) or 10% dialyzed HI-FCS (12 kDa cut off) added to the perfusate. The albumin group showed a gradual loss of endothelial function and integrity, whereas smooth muscle agonist and myogenic responses were retained. No remodeling was observed. Vessels cultured in the presence of serum showed a progressive constriction. Smooth muscle responses and substance P-induced endothelium-dependent dilation were maintained. An inward remodeling of 17 +/- 4% in the HI-FCS group and 26 +/- 3% in the dialyzed HI-FCS group was found, while media cross-sectional areas were unchanged. These data show that pressurized resistance arteries can be maintained in culture for several days and undergo eutrophic remodeling in vitro in the presence of high molecular weight serum factors.