Extended Culture Periods Research Articles

Electrospun 3D multi-scale fibrous scaffold for enhanced human dermal fibroblasts infiltration

Electrospun polymeric nanofibrous scaffold possesses significant potential in the field of tissue engineering due to its extracellular matrix mimicking topographical features that modulate a variety of key cellular activities. However, traditional two-dimensional (2D) electrospun scaffolds are generally close-packed fiber mats which prohibit cell infiltration and proliferation. Consequently, the applications of electrospun scaffolds in regenerative medicine are limited. In this study, we detail the use of a needle collector to fabricate three-dimensional (3D) electrospun poly-ε-caprolactone (PCL) scaffolds with multi-scale fiber dimensions. The resultant pore size is 4 times larger than conventional 2D electrospun scaffolds with interweaving micro (3.3 ± 0.6 µm) and nano (240 ± 50 nm) fibers. The scaffold was surface modified by grafting with gelatin molecules. It was found that surface modification significantly improved human dermal fibroblasts (HDFs) cell infiltration throughout the 3D multi-scale scaffold. Even after an extended culture period of up to 28 days, cell proliferation was well supported in the surface-modified 3D multi-scale scaffold as confirmed by Ki67 staining. Extracellular matrix proteins secreted by the HDFs was evident on the 3D multi-scale PCL scaffold showing promising potential to facilitate tissue regeneration, in particular dermal tissue engineering.

Abstract 4226: Human-derived hydrogels for 3D models of bone and osteosarcoma organoids and adipose tissue interactions

Abstract The goal of the study was to determine the metabolic and morphological changes, via high Content Imaging, in 3D models of osteosarcoma cancer cells using human derived hydrogels with different protein composition, Obagel and Obagel ECM. Furthermore, we evaluated the relevance of the adipose tissue microenvironment in Adipose Derived Stem Cells (ADSC) linage and Tumoroids development. Methods: Adipose derived Stem cells were cultured in a modified 3D human and fat and bone linage differentiation as studied via histology and Immunofluorescence. Osteosarcoma spheroids were cultured in Obatala Sciences’ human-derived hydrogels ObaGel and ObaGelECM and control media. Organoid structure and phenotypic changes were analyzed via live cell imaging on the Incucyte S3 and Nikon high content imaging device. Then, adipocyte- cancer cell crosstalk was evaluated using pooled adipocytes co-cultured with KHOS spheroids. Adipose derived Stem cells were cultured in a modified 3D human derived hydrogel dictates that promotes the differentiation potential toward fat and adipose tissue simultaneously in microenvironments within the organoid. Furthermore, the results demonstrated that ObaGel and ObaGel-ECM 3D cultures can support the growth and proliferation of KHOS tumor spheres during an extended culture period. Metabolic changes and phenotypic changes associated to ObaGel and ObaGel-ECM differentially facilitated tumoroids growth and migratory behavior. Conclusions: Human derived hydrogels are developed to support 3D culture of Bone and Osteosarcoma cells to recapitulate the phenotypic changes associated with their metastatic potential. The use of human derived 3D culture systems can accelerate the understanding of the role of the microenvironment in bone development and the pathogenesis and progression of Cancers like Osteosarcoma as well as screening for drug development. Citation Format: Cecilia G. Sanchez, Arman Raz, Haley Lassiter, Trivia Frazier. Human-derived hydrogels for 3D models of bone and osteosarcoma organoids and adipose tissue interactions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4226.

Abstract 4227: Human-derived hydrogels to support phenotypic changes in ER+ breast cancer cell line

Abstract Adipose tissue plays a critical role in breast cancer incidence, progression, and response to therapy. The development of human derived 3D culture systems can accelerate the understanding of the role of environmental factors in the progression of Breast cancer as well as a model for preclinical studies during drug development. MCF-7 spheroids were cultured in Obatala Sciences’ Obavate and human-derived hydrogels ObaGel® and ObaGel®-ECM and control media. Organoid structure and phenotypic changes were analyzed via live cell imaging on the Incucyte S3. Migration studies were performed and gene expression analysis of EMT markers. Finally, adipocyte-breast cancer cell crosstalk was evaluated using pooled adipocytes co-cultured with MCF-7 spheroids. The results demonstrated that ObaGel® and ObaGel®-ECM 3D cultures support the growth and proliferation of MCF-7 tumorspheres during an extended culture period. In addition, MCF-7 cells exhibit the characteristic morphology of tumorsphere-forming cells when cultured in Obavate, with morphological changes in the ObaGel® and ObaGel®-ECM 3D. ObaGel® and ObaGel®-ECM differentially supports MCF-7 migratory behavior and phenotypic changes characteristic of EMT. Conclusions: Human derived hydrogels are developed to support 3D culture of breast cancer cells and to recapitulate the phenotypic changes associated with their metastatic potential. Furthermore, the use of a human derived 3D coculture system provides a platform for the understanding of adipose tissue-breast cancer cell interactions as it relates to breast cancer initiation, progression, and treatment response. Defining these interactions will support the development of a tool for future use in patient stratification and precision medicine in identifying underlying causes of breast cancer progression and response to treatments. Citation Format: Cecilia G. Sanchez, Katie Hamel, Emma Rogers, Jordan Robinson, Haley Lassiter, Trivia Frazier, Christopher Williams. Human-derived hydrogels to support phenotypic changes in ER+ breast cancer cell line [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4227.

The effect of an extended culture period on birth weight among singletons born after single or double vitrified embryo transfer.

To evaluate the effect of an extended culture period on birth weight among singletons born after vitrified-warmed embryo transfer. A retrospective cohort study was performed among 12400 women who gave birth to 1015, 1027, 687, and 9671 singletons after single blastocyst transfer, single cleavage-stage embryo transfer, double blastocyst transfer, and double cleavage-stage embryo transfer, respectively. The unadjusted birth weight of singletons born after vitrified blastocyst transfer were heavier than those born after cleavage-stage transfer (β=30.28, SE=13.17, P=0.022), as were the adjusted birth weights (β=0.09, SE=0.03, P=0.007). In addition, there was a 37% increased odd of having an infant with high birth weight after vitrified blastocyst transfer compared with vitrified cleavage stage transfer (OR=1.37, 95% CI:1.07-1.77). The unadjusted and adjusted birth weight and odds of having an infant with high birth weight significantly increased after blastocyst transfer compared with cleavage-stage embryo transfer in vitrified-warmed cycles.

Distinguishing sterile inflammation from graft infection

We describe the case of a 68-year-old man who underwent ascending aortic replacement and thoracic endovascular aortic repair. Four years later, the patient developed neck pain on the right side and chest computed tomography showed expansion of fluid in the mediastinum which had extended to the neck. Echocardiography revealed advanced severity of aortic regurgitation and decreased ejection fraction. Given the progression of aortic regurgitation, decreased cardiac function, and rapidly expanding fluid accumulation causing neck pain, reoperation was indicated. All microbiological test including polymerase chain reaction were negative indicating absence of any infection. The patient is being followed-up without antibiotics and CT has not shown peri-graft fluid 2 years postoperatively. Since infection cannot be excluded completely, it is important to assess the condition with selective medium, extended culture periods, genetic testing, and consultations with microbiology laboratories when normal culture tests for general bacteria, and fungi are negative which can help avoid drug-resistant bacteria count, elevated medical costs, and drug side effects due to the improper use of antibiotics through proper diagnosis.

A Robust In Vitro Co-culture Model for Studying the Intercellular Communication of Neutrophils.

Communication among neutrophils plays critical roles during various phases of inflammatory responses, with clinical relevance to both acute and chronic inflammatory diseases. Despite its significance, underlying mechanisms are not well understood, due to the lack of an effective in vitro system to properly address this important question. Here we report a robust in vitro method to culture primary murine neutrophils derived from bone marrow, amenable for well-controlled studies of both neutrophil activation and intercellular communication among co-cultured neutrophils. This protocol can generate primary neutrophils with high purity and survival for an extended culture period, suitable for further phenotypic and functional analyses.

Long-term in vitro culture of 3D brain tissue model based on chitosan thermogel

Methods for studying brain function and disease heavily rely on in vivo animal models, ex-vivo tissue slices, and 2D cell culture platforms. These methods all have limitations that significantly impact the clinical translatability of results. Consequently, models able to better recapitulate some aspects of in vivo human brain are needed as additional preclinical tools. In this context, 3D hydrogel-based in vitro models of the brain are considered promising tools. To create a 3D brain-on-a-chip model, a hydrogel capable of sustaining neuronal maturation over extended culture periods is required. Among biopolymeric hydrogels, chitosan-β-glycerophosphate (CHITO-β-GP) thermogels have demonstrated their versatility and applicability in the biomedical field over the years. In this study, we investigated the ability of this thermogel to encapsulate neuronal cells and support the functional maturation of a 3D neuronal network in long-term cultures. To the best of our knowledge, we demonstrated for the first time that CHITO-β-GP thermogel possesses optimal characteristics for promoting neuronal growth and the development of an electrophysiologically functional neuronal network derived from both primary rat neurons and neurons differentiated from human induced pluripotent stem cells (h-iPSCs) co-cultured with astrocytes. Specifically, two different formulations were firstly characterized by rheological, mechanical and injectability tests. Primary nervous cells and neurons differentiated from h-iPSCs were embedded into the two thermogel formulations. The 3D cultures were then deeply characterized by immunocytochemistry, confocal microscopy, and electrophysiological recordings, employing both 2D and 3D micro-electrode arrays. The thermogels supported the long-term culture of neuronal networks for up to 100 d. In conclusion, CHITO-β-GP thermogels exhibit excellent mechanical properties, stability over time under culture conditions, and bioactivity toward nervous cells. Therefore, they are excellent candidates as artificial extracellular matrices in brain-on-a-chip models, with applications in neurodegenerative disease modeling, drug screening, and neurotoxicity evaluation.

Development of an ex vivo porcine skin model for the preclinical evaluation of subcutaneously injected biomacromolecules

Subcutaneous administration is used to deliver systemically-acting biotherapeutics, e.g. antibodies, and locally-acting biomacromolecules, e.g. hyaluronic acid. However, few preclinical models are available to evaluate post-injection behaviour in the tissue microenvironment. In vivo animal studies are costly, time-consuming, and raise obvious ethical concerns. In vitro models are cost-efficient, high-throughput solutions, but cannot simulate complex skin structure and biological function. An ex vivo model (containing hypodermis) with an extended culture period that enabled longitudinal studies would be of great interest for both the pharmaceutical and cosmeceutical industries. We describe the development of one such ex vivo model, using viable full-thickness porcine skin. Structural integrity was evaluated using a histological scoring system: spongiosis and epidermal detachment were identified as discriminating parameters. Ki67 and Claudin-1 expression reported on epidermal cell proliferation and barrier function, respectively and their expression decreased as a function of incubation time. After optimization, the system was used to investigate the fate/impact of subcutaneously administered hyaluronic acid (HA) formulations. The results showed that HA was localized at the injection site and adjacent adipocytes were well preserved during 5 days’ incubation and confirmed that the full-thickness ex vivo porcine skin model could provide a platform for preclinical evaluation of subcutaneously injected biomacromolecules.

Liver Extracellular Matrix-Based Nanofiber Scaffolds for the Culture of Primary Hepatocytes and Drug Screening.

Immortalized liver cell lines and primary hepatocytes are currently used as in vitro models for hepatotoxic drug screening. However, a decline in the viability and functionality of hepatocytes with time is an important limitation of these culture models. Advancements in tissue engineering techniques have allowed us to overcome this challenge by designing suitable scaffolds for maintaining viable and functional primary hepatocytes for a longer period of time in culture. In the current study, we fabricated liver-specific nanofiber scaffolds with polylactic acid (PLA) along with a decellularized liver extracellular matrix (LEM) by the electrospinning technique. The fabricated hybrid PLA-LEM scaffolds were more hydrophilic and had better swelling properties than the PLA scaffolds. The hybrid scaffolds had a pore size of 38 ± 8 μm and supported primary rat hepatocyte cultures for 10 days. Increased viability (2-fold increase in the number of live cells) and functionality (5-fold increase in albumin secretion) were observed in primary hepatocytes cultured on the PLA-LEM scaffolds as compared to those on conventional collagen-coated plates on day 10 of culture. A significant increase in CYP1A2 enzyme activity was observed in hepatocytes cultured on PLA-LEM hybrid scaffolds in comparison to those on collagen upon induction with phenobarbital. Drugs like acetaminophen and rifampicin showed the highest toxicity in hepatocytes cultured on hybrid scaffolds. Also, the lethal dose of these drugs in rodents was accurately predicted as 1.6 g/kg and 594 mg/kg, respectively, from the corresponding IC50 values obtained from drug-treated hepatocytes on hybrid scaffolds. Thus, the fabricated liver-specific electrospun scaffolds maintained primary hepatocyte viability and functionality for an extended period in culture and served as an effective ex vivo drug screening platform to predict an accurate in vivo drug-induced hepatotoxicity.

Abstract P1127: Induced Pluripotent Stem Cell-derived Macrophages Modulate Human Engineered Cardiac Tissue Function

In the native human myocardium, cardiac resident macrophages are a critical mediator for normal cardiac function, performing a variety of biological roles in electrical conduction, regeneration, and phagocytosis and efferocytosis. However, most models of human engineered cardiac tissues (hECT), which aim to recapitulate the native adult myocardium for disease modeling and drug screening applications, do not include a macrophage population. Thus, in this study we introduced an iPSC-derived macrophage (iM0) population into an all iPSC-derived tissue engineered model of the human myocardium in order to assess its impact on tissue phenotype and function. CD14 + macrophages were differentiated from iPSCs and added as 7% of the total cell composition (7% iM0, 69.75% iCM, 23.25% iCF) in the hECT model. The hECTs were then cultured for 4 weeks and subjected to a ramped electrical stimulation protocol for matured function. The resulting tissues showed that the presence of a CD68 + macrophage population was maintained over this extended period of culture and significantly impacted hECT function, increasing contractile force production and modulating contraction and relaxation velocity. Interrogation of calcium signaling via isoproterenol revealed that iM0 presence in the tissues may be causing these functional differences via modulation of the cAMP signaling pathway. These observations demonstrate that macrophages significantly contribute to cardiac function in iPSC-derived hECT models and emphasize the need to further explore their contributions not only in healthy hECT models, but also in the contexts of disease and injury.

M-MDSC in vitro generation from mouse bone marrow with IL-3 reveals high expression and functional activity of arginase 1.

Myeloid-derived suppressor cells (MDSC) represent major regulators of immune responses, which can control T cells via their inducible nitric oxide synthase (iNOS)- and arginase 1 (Arg1)-mediated effector functions. While GM-CSF is well documented to promote MDSC development, little is known about this potential of IL-3, an established growth factor for mast cells. Here, we show that IL-3, similar to GM-CSF, generates monocytic MDSC (M-MDSC) from murine bone marrow (BM) cells after 3 days of in vitro culture. At this time point, predominantly CD11b+ CD49a+ monocytic and CD11b+ CD49a- FcεR I- neutrophilic cells were detectable, while CD11blow/neg FcεR I+ mast cells accumulated only after extended culture periods. Both growth factors were equivalent in generating M-MDSC with respect to phenotype, cell yield and typical surface markers. However, IL-3 generated M-MDSC produced less TNF, IL-1β and IL-10 after activation with LPS + IFN-γ but showed higher Arg1 expression compared to GM-CSF generated M-MDSC. Arg1 was further induced together with iNOS after MDSC activation. Accordingly, an increased Arg1-dependent suppressor activity by the IL-3 generated M-MDSC was observed using respective iNOS and Arg1 inhibitors. Together, these data indicate that M-MDSC can be generated in vitro by IL-3, similar to GM-CSF, but with increased Arg1 expression and Arg1-mediated suppression capacity. This protocol now allows further in vitro studies on the role of IL-3 for MDSC biology.

A cell wall-targeted organic-inorganic hybrid nano-catcher for ultrafast capture and SERS detection of invasive fungi

Due to the extended culture period and various inconveniences in vitro culture, the detection of invasive fungi is rather difficult, leading to high mortality rates of the diseases caused by them. It is, however, crucial for clinical therapy and lowering patient mortality to quickly identify invasive fungus from clinical specimens. A promising non-destructive method for finding fungi is surface-enhanced Raman scattering (SERS), however, its substrate has a low level of selectivity. Clinical sample components can obstruct the target fungi's SERS signal on account of their complexity. Herein, an MNP@PNIPAMAA hybrid organic-inorganic nano-catcher was created by using ultrasonic-initiated polymerization. The caspofungin (CAS), a fungus cell wall-targeting drug, is used in this study. We investigated MNP@PNIPAMAA-CAS as a technique to rapidly extract fungus from complex samples under 3 s. SERS could subsequently be used to instantly identify the fungi that were successfully isolated with an efficacy rate of about 75%. The entire process took just 10 min. This method is an important breakthrough that might be advantageous in terms of the rapid detection of invasive fungi.

Abstract 11681: Engineering of an IPSC Derived Perfusable and Thick Three-Layer Cardiac Tissue

Introduction: The left ventricular wall is an approximately 1 cm thick tissue composing of vascular networks and three distinct layers: the endocardium, the myocardium, and the epicardium. Engineering of a perfusable and thick three-layer cardiac tissue from human induced pluripotent stem cells (hiPSCs) has not been accomplished to date. Here, we describe formation of a perfusable and thick three-layer engineered cardiac tissue (PTTL-ECT) using additive and subtractive manufacturing. Methods: hiPSCs were differentiated into endocardial (Endo) cells, cardiomyocytes (CMs) and epicardial (Epi) cells. To create a perfusable vascular network in an engineered cardiac tissue, we used a water-soluble heat extruded material to print a sacrificial pattern that would eventually dissolve and become a vascular lumen. By casting hydrogels and the three types of cells derived from hiPSCs, we stacked the endocardial layer, myocardial layer, and epicardial layer mimicking the architecture of the ventricular wall . We then compared PTTL-ECT with engineered tissues without perfusable vascular channels (TTL-ECT) and single layer perfused tissues (PTSL-ECT). Results: Purity of hiPSC derived-Endo cells, -CMs, and -Epi cells reached 96.98±1.33% for NFATC1 + and CD31 + Endo-cells, 83.95±1.77% for cTNT + CMs, and 97.75±1.75% for WT1 + Epi-cells, respectively. The PTTL-ECT had 1.2 cm thickness at day 1 after casting. Histological analysis of the TTL-ECT without vascular channel showed a necrotic core with only the outermost cell layer survived after 4 weeks of culture. Addition of the vascular channel enabled cell survival across the thickness of the entire PTTL-ECT, while the three-layered structure remained intact over four weeks of perfusion culture. CMs in the PTTL-ECT showed enhanced maturity in terms of gene expression profiles, sarcomere lengths and contraction force compared with PTSL-ECT. Conclusions: By combining additive and subtractive manufacturing methods, we successfully generated a perfused, three layers thick cardiac graft that maintained architecture and viability over extended culture periods. The PTTL-ECT could be an useful in vitro model that mimic the native ventricular tissue in disease modeling and drug testing.

Highly porous multiple-cell-laden collagen/hydroxyapatite scaffolds for bone tissue engineering

Efficient vascularization within a scaffold is an essential criterion for evaluating the success of volumetric bone formation. Various strategies using angiogenic growth factors and cell-based approaches to induce effective osteogenic and angiogenic activities have been investigated. In this study, we propose a new highly porous multiple-cell-laden collagen/hydroxyapatite scaffold fabricated using a whipped bioink. After in vitro culturing of cells in the porous scaffolds for an extended culture period, osteogenic and angiogenic activities were significantly enhanced owing to the well-developed microporous cell-supporting matrix inducing efficient crosstalk between the adipose stem cells and endothelial cells compared to those of the normally bioprinted cell-constructs. Furthermore, the in vitro results were thoroughly evaluated by in vivo experiments using a posterolateral lumbar spinal fusion model of an ovariectomized mouse. Based on these results, the porous multiple-cell-laden scaffolds enhanced spine fusion in the event of osteoporosis.

Excessive Oxalic Acid Secreted by Sparassis latifolia Inhibits the Growth of Mycelia during Its Saprophytic Process.

Sparassis latifolia is an edible and medicinal mushroom in Asia commercially cultivated on substrates containing pine sawdust. Its slow mycelial growth rate greatly increases the cultivation cycle. In this study, we mainly studied the role of oxalic acid (OA) secreted by S. latifolia in its saprophytic process. Our results show that crystals observed on the mycelial surface contained calcium oxalate monohydrate (COM) and calcium oxalate dihydrate (COD) according to X-ray diffraction (XRD). Vegetative mycelia secreted large amounts of OA during extended culture periods. However, high concentrations of OA decreased the mycelial growth rate significantly. Moreover, the degradation of lignocellulose was significantly inhibited under high concentrations of OA. These changes could be attributed to the significantly decreased activities of lignocellulose-degrading enzymes. In conclusion, by establishing a link between OA secretion by the mycelium and the slow growth rate of its saprophytic process, this work provides fundamental information for shortening the cultivation cycle of S. latifolia.

Mesenchymal tumor organoid models recapitulate rhabdomyosarcoma subtypes

Rhabdomyosarcomas (RMS) are mesenchyme‐derived tumors and the most common childhood soft tissue sarcomas. Treatment is intense, with a nevertheless poor prognosis for high‐risk patients. Discovery of new therapies would benefit from additional preclinical models. Here, we describe the generation of a collection of 19 pediatric RMS tumor organoid (tumoroid) models (success rate of 41%) comprising all major subtypes. For aggressive tumors, tumoroid models can often be established within 4–8 weeks, indicating the feasibility of personalized drug screening. Molecular, genetic, and histological characterization show that the models closely resemble the original tumors, with genetic stability over extended culture periods of up to 6 months. Importantly, drug screening reflects established sensitivities and the models can be modified by CRISPR/Cas9 with TP53 knockout in an embryonal RMS model resulting in replicative stress drug sensitivity. Tumors of mesenchymal origin can therefore be used to generate organoid models, relevant for a variety of preclinical and clinical research questions.

Porcine choroid plexus-Riems cell line demonstrates altered polarization of transport proteins compared with the native epithelium.

The choroid plexus epithelium (CPe) forms a barrier between the cerebral blood supply and the cerebrospinal fluid (CSF), establishing the blood-CSF barrier (BCSFB). CSF is actively secreted by the CPe via tightly controlled processes involving multiple channels, transporters, and pumps. The importance of controlling CSF production and composition has been accentuated recently with an appreciation of CSF dysfunction in many pathologies. For mechanistic studies of CSF production, isolated CPe cell lines are valuable for the testing of hypotheses and potential drug targets. Although several continuous CPe cell lines have been described, none appear to have all the characteristics of the native epithelium and each must be used judiciously. The porcine choroid plexus-Riems (PCP-R) cell line forms a high-resistance monolayer characteristic of a barrier epithelium. Conservation of this phenotype is unusual among CPe cell lines, making this model useful for studies of the effects of infection, injury, and drugs on permeability. We have recently discovered that, although this line expresses many of the transporters expressed in the native tissue, some are mispolarized. As a result, inferences regarding fluid/electrolyte flux and the resultant CSF production should be pursued with caution. Furthermore, extended culture periods and changes in media composition result in significant morphological and functional variability. These studies provide a more detailed characterization of the PCP-R cell line concerning transporter expression, polarization, and functionality, as well as plasticity in culture, with the goal to provide the scientific community with information necessary to optimize future experiments with this model.

Hematopoietic Stem Cell-Independent Differentiation of Mast Cells From Mouse Intraembryonic VE-Cadherin+ Cells.

Hematopoietic stem cell (HSC)-independent hematopoiesis from hemogenic endothelial cells (HECs) in the mouse embryo has been recognized as a source of tissue-resident hematopoietic cells in adult mice. Connective tissue mast cells (MCs) have been reported to originate from VE-cadherin (VE-cad)-expressing HECs in the yolk sac and embryo proper (EP) by a VE-cad-Cre-mediated lineage-tracing analysis. However, it remains unclear whether MCs are generated via a conventional HSC-dependent hematopoietic differentiation pathway, or whether through a fast-track pathway bypassing the emergence of HSCs. Here, we investigated whether EP-derived VE-cad+ cells differentiate into MCs independently of HSCs. VE-cad+ cells isolated from the embryonic day (E) 9.5-10.5 EP robustly formed connective tissue-type MCs in a newly established co-culture system using PA6 stromal cells. In contrast, bone marrow (BM) reconstitution assays of cultured cells indicated that E9.5 VE-cad+ cells did not differentiate into transplantable HSCs in this culture condition. Lymphoid-biased HSCs with a limited self-renewal capacity were occasionally detected in some cultures of E10.5 VE-cad+ cells, while MC growth was constantly observed in all cultures examined. HSCs purified from adult BM required a more extended culture period to form MCs in the PA6 co-culture than the embryonic VE-cad+ cells. Furthermore, E9.5-E10.5 VE-cad+ cells contributed to tissue-resident MCs in postnatal mice when transplanted into the peritoneal cavity of newborn mice. These results suggest that EP-derived VE-cad+ cells generate MCs independently of HSC development in vitro and possess the potential of generating connective tissue MCs in vivo, although the exact differentiation program remains unsolved.

Why Is It So Difficult To Have Competent Oocytes from In vitro Cultured Preantral Follicles?

The developmental competence of oocytes is acquired gradually during follicular development, mainly through oocyte accumulation of RNA molecules and proteins that will be used during fertilization and early embryonic development. Several attempts to develop in vitro culture systems to support preantral follicle development up to maturation are reported in the literature, but oocyte competence has not yet been achieved in human and domestic animals. The difficulties to have fertilizable oocytes are related to thousands of mRNAs and proteins that need to be synthesized, long-term duration of follicular development, size of preovulatory follicles, composition of in vitro culture medium, and the need of multi-step culture systems. The development of a culture system that maintains bidirectional communication between the oocyte and granulosa cells and that meets the metabolic demands of each stage of follicle growth is the key to sustain an extended culture period. This review discusses the physiological and molecular mechanisms that determine acquisition of oocyte competence in vitro, like oocyte transcriptional activity, follicle and oocyte sizes, and length and regulation of follicular development in murine, human, and domestic animal species. The state of art of in vitro follicular development and the challenges to have complete follicular development in vitro are also highlighted.

Adipose-derived cells: building blocks of three-dimensional microphysiological systems.

ABSTRACTMicrophysiological systems (MPS) created with human-derived cells and biomaterial scaffolds offer a potential in vitro alternative to in vivo animal models. The adoption of three-dimensional MPS models has economic, ethical, regulatory, and scientific implications for the fields of regenerative medicine, metabolism/obesity, oncology, and pharmaceutical drug discovery. Key opinion leaders acknowledge that MPS tools are uniquely positioned to aid in the objective to reduce, refine, and eventually replace animal experimentation while improving the accuracy of the finding’s clinical translation. Adipose tissue has proven to be an accessible and available source of human-derived stromal vascular fraction (SVF) cells, a heterogeneous population available at point of care, and adipose-derived stromal/stem cells, a relatively homogeneous population requiring plastic adherence and culture expansion of the SVF cells. The adipose-derived stromal/stem cells or SVF cells, in combination with human tissue or synthetic biomaterial scaffolds, can be maintained for extended culture periods as three-dimensional MPS models under angiogenic, stromal, adipogenic, or osteogenic conditions. This review highlights recent literature relating to the versatile use of adipose-derived cells as fundamental components of three-dimensional MPS models for discovery research and development. In this context, it compares the merits and limitations of the adipose-derived stromal/stem cells relative to SVF cell models and considers the likely directions that this emerging field of scientific discovery will take in the near future.