Transcriptomic, epigenomic, and spatial metabolomic cell profiling redefines regional human kidney anatomy.

A large-scale multimodal atlas that includes major kidney regions is lacking. Here, we employed simultaneous high-throughput single-cell ATAC/RNA sequencing (SHARE-seq) and spatially resolved metabolomics to profile 54 human samples from distinct kidney anatomical regions. We generated transcriptomes of 446,267 cells and chromatin accessibility profiles of 401,875 cells and developed a package to analyze 408,218 spatially resolved metabolomes. We find that the same cell type, including thin limb, thick ascending limb loop of Henle and principal cells, display distinct transcriptomic, chromatin accessibility, and metabolomic signatures, depending on anatomic location. Surveying metabolism-associated gene profiles revealed non-overlapping metabolic signatures between nephron segments and dysregulated lipid metabolism in diseased proximal tubule (PT) cells. Integrating multimodal omics with clinical data identified PLEKHA1 as a disease marker, and its in vitro knockdown increased gene expression in PT differentiation, suggesting possible pathogenic roles. This study highlights previously underrepresented cellular heterogeneity underlying the human kidney anatomy.

Predicting proximal tubule failed repair drivers through regularized regression analysis of single cell multiomic sequencing.

Renal proximal tubule epithelial cells have considerable intrinsic repair capacity following injury. However, a fraction of injured proximal tubule cells fails to undergo normal repair and assumes a proinflammatory and profibrotic phenotype that may promote fibrosis and chronic kidney disease. The healthy to failed repair change is marked by cell state-specific transcriptomic and epigenomic changes. Single nucleus joint RNA- and ATAC-seq sequencing offers an opportunity to study the gene regulatory networks underpinning these changes in order to identify key regulatory drivers. We develop a regularized regression approach to construct genome-wide parametric gene regulatory networks using multiomic datasets. We generate a single nucleus multiomic dataset from seven adult human kidney samples and apply our method to study drivers of a failed injury response associated with kidney disease. We demonstrate that our approach is a highly effective tool for predicting key cis- and trans-regulatory elements underpinning the healthy to failed repair transition and use it to identify NFAT5 as a driver of the maladaptive proximal tubule state.

Decellularized human pancreatic extracellular matrix-based physiomimetic microenvironment for human islet culture.

A strategy that seeks to combine the biophysical properties of inert encapsulation materials like alginate with the biochemical niche provided by pancreatic extracellular matrix (ECM)-derived biomaterials, could provide a physiomimetic pancreatic microenvironment for maintaining long-term islet viability and function in culture. Herein, we have demonstrated that incorporating human pancreatic decellularized ECM within alginate microcapsules results in a significant increase in Glucose Stimulation Index (GSI) and total insulin secreted by encapsulated human islets, compared to free islets and islets encapsulated in only alginate. ECM supplementation also resulted in long-term (58 days) maintenance of GSI levels, similar to that observed in free islets at the first time point (day 5). At early time points in culture, ECM promoted gene expression changes through ECM- and cell adhesion-mediated pathways, while it demonstrated a mitochondria-protective effect in the long-term. STATEMENT OF SIGNIFICANCE: The islet isolation process can damage the islet extracellular matrix, resulting in loss of viability and function. We have recently developed a detergent-free, DI-water based method for decellularization of human pancreas to produce a potent solubilized ECM. This ECM was added to alginate for microencapsulation of human islets, which resulted in significantly higher stimulation index and total insulin production, compared to only alginate capsules and free islets, over long-term culture. Using ECM to preserve islet health and function can improve transplantation outcomes, as well as provide novel materials and platforms for studying islet biology in microfluidic, organ-on-a-chip, bioreactor and 3D bioprinted systems.

Mitochondria Transplantation Mitigates Damage in an In Vitro Model of Renal Tubular Injury and in an Ex Vivo Model of DCD Renal Transplantation.

OBJECTIVES: To test whether mitochondrial transplantation (MITO) mitigates damage in 2 models of acute kidney injury (AKI). BACKGROUND: MITO is a process where exogenous isolated mitochondria are taken up by cells. As virtually any morbid clinical condition is characterized by mitochondrial distress, MITO may find a role as a treatment modality in numerous clinical scenarios including AKI. METHODS: For the in vitro experiments, human proximal tubular cells were damaged and then treated with mitochondria or placebo. For the ex vivo experiments, we developed a non-survival ex vivo porcine model mimicking the donation after cardiac death renal transplantation scenario. One kidney was treated with mitochondria, although the mate organ received placebo, before being perfused at room temperature for 24 hours. Perfusate samples were collected at different time points and analyzed with Raman spectroscopy. Biopsies taken at baseline and 24 hours were analyzed with standard pathology, immunohistochemistry, and RNA sequencing analysis. RESULTS: In vitro, cells treated with MITO showed higher proliferative capacity and adenosine 5'-triphosphate production, preservation of physiological polarization of the organelles and lower toxicity and reactive oxygen species production. Ex vivo, kidneys treated with MITO shed fewer molecular species, indicating stability. In these kidneys, pathology showed less damage whereas RNAseq analysis showed modulation of genes and pathways most consistent with mitochondrial biogenesis and energy metabolism and downregulation of genes involved in neutrophil recruitment, including IL1A, CXCL8, and PIK3R1. CONCLUSIONS: MITO mitigates AKI both in vitro and ex vivo.

Regenerative medicine technologies applied to transplant medicine. An update.

Regenerative medicine (RM) is changing how we think and practice transplant medicine. In regenerative medicine, the aim is to develop and employ methods to regenerate, restore or replace damaged/diseased tissues or organs. Regenerative medicine investigates using tools such as novel technologies or techniques, extracellular vesicles, cell-based therapies, and tissue-engineered constructs to design effective patient-specific treatments. This review illustrates current advancements in regenerative medicine that may pertain to transplant medicine. We highlight progress made and various tools designed and employed specifically for each tissue or organ, such as the kidney, heart, liver, lung, vasculature, gastrointestinal tract, and pancreas. By combing both fields of transplant and regenerative medicine, we can harbor a successful collaboration that would be beneficial and efficacious for the repair and design of de novo engineered whole organs for transplantations.

Bioengineering of the digestive tract: approaching the clinic.

The field of regenerative medicine is developing technologies that, in the near future, will offer alternative approaches to either cure diseases affecting the gastrointestinal tract or slow their progression by leveraging the intrinsic ability of our tissues and organs to repair after damage. This article will succinctly illustrate the three technologies that are closer to clinical translation-namely, human intestinal organoids, sphincter bioengineering and decellularization, whereby the cellular compartment of a given segment of the digestive tract is removed to obtain a scaffold consisting of the extracellular matrix. The latter will be used as a template for the regeneration of a functional organ, whereby the newly generated cellular compartment will be obtained from the patient's own cells. Although clinical application of this technology is approaching, product development challenges are being tackled to warrant safety and efficacy.

Comprehensive characterization of the human pancreatic proteome for bioengineering applications.

Interactions between the pancreatic extracellular matrix (ECM) and islet cells are known to regulate multiple aspects of islet physiology, including survival, proliferation, and glucose-stimulated insulin secretion. Recognizing the essential role of ECM in islet survival and function, various engineering approaches have been developed that aim to utilize ECM-based materials to recreate a native-like microenvironment. However, a major impediment to the success of these approaches has been the lack of a robust and comprehensive characterization of the human pancreatic proteome. Herein, by combining mass spectrometry (MS) and multiplex ELISA, we have provided an improved workflow for the in-depth profiling of the proteome, including minor constituents that are generally underrepresented. Moreover, we have further validated the effectiveness of our detergent-free decellularization protocol in the removal of cellular proteins and retention of the matrisome. It has also been established that the decellularized ECM and its derivatives can provide more tissue-specific cues than traditionally used biological scaffolds and are therefore more physiologically relevant for the development of hydrogels, bioinks and medium additives, in order to create a pancreatic niche. The data generated in this study would contribute significantly to the efforts of comprehensively defining the ECM atlas and also serve as a standard for the human pancreatic proteome to provide further guidance for design and engineering strategies for improved tissue engineering scaffolds.

Spheroid Trapping and Calcium Spike Estimation Techniques toward Automation of 3D Culture.

We present a spheroid trapping device, compatible with traditional tissue culture plates, to confine microtissues in a small area and allow suspension cultures to be treated like adherent cultures with minimal loss of spheroids due to aspiration. We also illustrate an automated morphology-independent procedure for cell recognition, segmentation, and a calcium spike detection technique for high-throughput analysis in 3D cultured tissue. Our cell recognition technique uses a maximum intensity projection of spatial-temporal data to create a binary mask, which delineates individual cell boundaries and extracts mean fluorescent data for each cell through a series of intensity thresholding and cluster labeling operations. The temporal data are subject to sorting for imaging artifacts, baseline correction, smoothing, and spike detection algorithms. We validated this procedure through analysis of calcium data from 2D and 3D SHSY-5Y cell cultures. Using this approach, we rapidly created regions of interest (ROIs) and extracted fluorescent intensity data from hundreds of cells in the field of view with superior data fidelity over hand-drawn ROIs even in dense (3D tissue) cell populations. We sorted data from cells with imaging artifacts (such as photo bleaching and dye saturation), classified nonfiring and firing cells, estimated the number of spikes in each cell, and documented the results, facilitating large-scale calcium imaging analysis in both 2D and 3D cultures. Since our recognition and segmentation technique is independent of morphology, our protocol provides a versatile platform for the analysis of large confocal calcium imaging data from neuronal cells, glial cells, and other cell types.

Detergent-Free Decellularization of the Human Pancreas for Soluble Extracellular Matrix (ECM) Production.

Islet transplantation (ITx) has the potential to become the standard of care in beta cell replacement medicine but its results remain inferior to those obtained with whole pancreas transplantation. The protocols currently used for human islet isolation are under scrutiny because they are based on the enzymatic digestion of the organ, whereby the pancreas is demolished, its connections to the body are lost and islets are irreversibly damaged. Islet damage is characterized by critical factors such as the destruction of the extracellular matrix (ECM), which represents the 3D framework of the islet niche and whose loss is incompatible with islet euphysiology. Researchers are proposing the use of ECM-based scaffolds derived from the mammalian pancreas to address this problem and ultimately improve islet viability, function, and lifespan. Currently available methods to obtain such scaffolds are harsh because they are largely detergent based. Thus, we propose a new, detergent-free method that creates less ECM damage and can preserve critical components of pancreatic ECM. The results show that the newly developed decellularization protocol allowed the achievement of complete DNA clearance while the ECM components were retained. The ECM obtained was tested for cytotoxicity and encapsulated with human pancreatic islets which showed a positive cellular behavior with insulin secretion when stimulated with glucose challenge. Collectively, we propose a new method for the decellularization of the human pancreas without the use of conventional ionic and non-ionic chemical detergents. This protocol and the ECM obtained with it could be of use for both in vitro and in vivo applications.

Secretome-Based Prediction of Three-Dimensional Hepatic Microtissue Physiological Relevance.

Early biomarkers for indication of the complex physiological relevance (CPR) of a three-dimensional (3D) tissue model are needed. CPR is detected late in culture and requires different analytical techniques. Albumin production, CYP3A4 expression, and formation of bile canaliculi structures are commonly used to compare in vitro hepatic cells to their in vivo counterpart. A universal biomarker independent of the cell type would bring this to a common detection platform. We make the case that these hepatic characteristics are not sufficient to differentiate traditional (2D) cell culture from the more complex 3D culture. We explored the cytokine secretion profile (secretome) for its potential as a 3D early culture biomarker. PDGF-AB/BB and vascular endothelial growth factor (VEGF) were found to be upregulated in 3D compared to 2D cultures at early time points (days 3 and 4). These observations provide a foundation upon which in vivo validation of cytokines can lead to physiologically relevant 3D in vitro cell culture.

Calcium Oscillation Frequency Is a Potential Functional Complex Physiological Relevance Indicator for a Neuroblastoma-Based 3D Culture Model.

In vitro screening for drugs that affect neural function in vivo is still primitive. It primarily relies on single cellular responses from 2D monolayer cultures that have been shown to be exaggerations of the in vivo response. For the 3D model to be physiologically relevant, it should express characteristics that not only differentiate it from 2D but also closely emulate those seen in vivo. These complex physiologically relevant (CPR) outcomes can serve as a standard for determining how close a 3D culture is to its native tissue or which out of a given number of 3D platforms is better suited for a given application. In this study, Fluo-4-based calcium fluorescence imaging was performed followed by automated image data processing to quantify the calcium oscillation frequency of SHSY5Y cells cultured in 2D and 3D formats. It was found that the calcium oscillation frequency is upregulated in traditional 2D cultures while it was comparable to in vivo in spheroid and microporous polymer scaffold-based 3D models, suggesting calcium oscillation frequency as a potential functional CPR indicator for neural cultures.

Extracellular matrix-based hydrogels obtained from human tissues: a work still in progress.

PURPOSE OF REVIEW: The current review summarizes contemporary decellularization and hydrogel manufacturing strategies in the field of tissue engineering and regenerative medicine. RECENT FINDINGS: Decellularized extracellular matrix (ECM) bioscaffolds are a valuable biomaterial that can be purposed into various forms of synthetic tissues such as hydrogels. ECM-based hydrogels can be of animal or human origin. The use of human tissues as a source for ECM hydrogels in the clinical setting is still in its infancy and current literature is scant and anecdotal, resulting in inconclusive results. SUMMARY: Thus far the methods used to obtain hydrogels from human tissues remains a work in progress. Gelation, the most complex technique in obtaining hydrogels, is challenging due to remarkable heterogeneity of the tissues secondary to interindividual variability. Age, sex, ethnicity, and preexisting conditions are factors that dramatically undermine the technical feasibility of the gelation process. This is contrasted with animals whose well defined anatomical and histological characteristics have been selectively bred for the goal of manufacturing hydrogels.

Utility of extracellular matrix powders in tissue engineering.

Extracellular matrix (ECM) materials have had remarkable success as scaffolds in tissue engineering (TE) and as therapies for tissue injury whereby the ECM microenvironment promotes constructive remodeling and tissue regeneration. ECM powder and solubilized derivatives thereof have novel applications in TE and RM afforded by the capacity of these constructs to be dynamically modulated. The powder form allows for effective incorporation and penetration of reagents; hence, ECM powder is an efficacious platform for 3D cell culture and vehicle for small molecule delivery. ECM powder offers minimally invasive therapy for tissue injury and successfully treatment for wounds refractory to first-line therapies. Comminution of ECM and fabrication of powder-derived constructs, however, may compromise the biological integrity of the ECM. The current lack of optimized fabrication protocols prevents a more extensive and effective clinical application of ECM powders. Further study on methods of ECM powder fabrication and modification is needed.

Evaluation of cellular adhesion and organization in different microporous polymeric scaffolds.

The lack of prediction accuracy during drug development and screening risks complications during human trials, such as drug-induced liver injury (DILI), and has led to a demand for robust, human cell-based, in vitro assays for drug discovery. Microporous polymer-based scaffolds offer an alternative to the gold standard flat tissue culture plastic (2D TCPS) and other 3D cell culture platforms as the porous material entraps cells, making it advantageous for automated liquid handlers and high-throughput screening (HTS). In this study, we optimized the surface treatment, pore size, and choice of scaffold material with respect to cellular adhesion, tissue organization, and expression of complex physiologically relevant (CPR) outcomes such as the presence of bile canaliculi-like structures. Poly-l-lysine and fibronectin (FN) coatings have been shown to encourage cell attachment to the underlying substrate. Treatment of the scaffold surface with NaOH followed with a coating of FN improved cell attachment and penetration into pores. Of the two pore sizes we investigated (A: 104 ± 4 µm; B: 175 ± 6 µm), the larger pore size better promoted cell penetration while limiting tissue growth from reaching the hypoxia threshold. Finally, polystyrene (PS) proved to be conducive to cell growth, penetration into the scaffold, and yielded CPR outcomes while being a cost-effective choice for HTS applications. These observations provide a foundation for optimizing microporous polymer-based scaffolds suitable for drug discovery. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:505-514, 2018.

3D nerve cell cultures and complex physiological relevance.

The field of tissue engineering has not yet provided knowledge on which a consensus for the complex physiological relevance (CPR) of neuronal cultures could be established. The CPR of 3D neuronal cultures can have a profound impact on the drug discovery process through the validation of in vitro models for the study of neuropsychiatric and degenerative diseases, as well as screening for neurotoxicity during drug development. Herein, we assemble evidence in support of the potential of [Ca2+]i oscillation frequency as a CPR outcome that can demonstrate the in vivo-like behavior of 3D cultures and differentiate them from 2D monolayers. We demonstrate that [Ca2+]i oscillation frequencies in 2D cultures are significantly higher than those found in 3D cultures, and provide a possible molecular explanation.

Molecular basis for cytokine biomarkers of complex 3D microtissue physiology in vitro.

'Physiologically more-relevant' claims are readily made for cells cultured on any surface or in a scaffold that provides loosely defined 3D geometry. A set of tools to measure culture '3D-ness' more accurately are needed. Such tools should find applications in fields ranging from high-throughput identification of substrates for tissue engineering and regenerative medicine to cell-based screening of drug candidates. Until now, these fields have not provided a consensus for the most promising place to initiate the search. Here, we review recent advances in transcriptomic, proteomic, inflammation and oncology-related pathways, as well as functional studies that strongly point to cytokines as the most likely compounds to form the missing consensus.

Is time an extra dimension in 3D cell culture?

Time or the temporal microenvironment is a parameter that is often overlooked in 3D cell culture. However, given that the 3D system is a dynamic entity, there exists bidirectional signaling between the cells and their microenvironment and, in time, cells can develop the capacity to modulate their environment. We make this case here by illustrating the relation between the temporal dimension and other microenvironmental parameters and demonstrate how the exogenously incorporated microenvironmental factors (MEFs) can be rendered less significant with time. Such knowledge can guide construct design to make 3D platforms architecturally simpler by eliminating redundancy. We further show that there is a need to establish the point at which the construct is complex enough such that its use yields responses that more closely emulate in vivo outcomes.

Biophysical microenvironment and 3D culture physiological relevance.

Force and substrate physical property (pliability) is one of three well established microenvironmental factors (MEFs) that may contribute to the formation of physiologically more relevant constructs (or not) for cell-based high-throughput screening (HTS) in preclinical drug discovery. In 3D cultures, studies of the physiological relevance dependence on material pliability are inconclusive, raising questions regarding the need to design platforms with materials whose pliability lies within the physiological range. To provide more insight into this question, we examine the factors that may underlie the studies inconclusiveness and suggest the elimination of redundant physical cues, where applicable, to better control other MEFs, make it easier to incorporate 3D cultures into state of the art HTS instrumentation, and reduce screening costs per compound.

Microtissue size and hypoxia in HTS with 3D cultures.

The three microenvironmental factors that characterize 3D cultures include: first, chemical and/or biochemical composition, second, spatial and temporal dimensions, and third, force and/or substrate physical properties. Although these factors have been studied individually, their interdependence and synergistic interactions have not been well appreciated. We make this case by illustrating how microtissue size (spatial) and hypoxia (chemical) can be used in the formation of physiologically more relevant constructs (or not) for cell-based high-throughput screening (HTS) in drug discovery. We further show how transcriptomic and/or proteomic results from heterogeneously sized microtissues and scaffold architectures that deliberately control hypoxia can misrepresent and represent in vivo conditions, respectively. We offer guidance, depending on HTS objectives, for rational 3D culture platform choice for better emulation of in vivo conditions.

Neural cell 3D microtissue formation is marked by cytokines' up-regulation.

Cells cultured in three dimensional (3D) scaffolds as opposed to traditional two-dimensional (2D) substrates have been considered more physiologically relevant based on their superior ability to emulate the in vivo environment. Combined with stem cell technology, 3D cell cultures can provide a promising alternative for use in cell-based assays or biosensors in non-clinical drug discovery studies. To advance 3D culture technology, a case has been made for identifying and validating three-dimensionality biomarkers. With this goal in mind, we conducted a transcriptomic expression comparison among neural progenitor cells cultured on 2D substrates, 3D porous polystyrene scaffolds, and as 3D neurospheres (in vivo surrogate). Up-regulation of cytokines as a group in 3D and neurospheres was observed. A group of 13 cytokines were commonly up-regulated in cells cultured in polystyrene scaffolds and neurospheres, suggesting potential for any or a combination from this list to serve as three-dimensionality biomarkers. These results are supportive of further cytokine identification and validation studies with cells from non-neural tissue.