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PloS One 2020A lack of perfusion has been one of the most significant obstacles for three-dimensional culture systems of organoids and embryonic tissues. Here, we developed a simple...
A lack of perfusion has been one of the most significant obstacles for three-dimensional culture systems of organoids and embryonic tissues. Here, we developed a simple and reliable method to implement a perfusable capillary network in vitro. The method employed the self-organization of endothelial cells to generate a capillary network and a static pressure difference for culture medium circulation, which can be easily introduced to standard biological laboratories and enables long-term cultivation of vascular structures. Using this culture system, we perfused the lumen of the self-organized capillary network and observed a flow-induced vascular remodeling process, cell shape changes, and collective cell migration. We also observed an increase in cell proliferation around the self-organized vasculature induced by flow, indicating functional perfusion of the culture medium. We also reconstructed extravasation of tumor and inflammatory cells, and circulation inside spheroids including endothelial cells and human lung fibroblasts. In conclusion, this system is a promising tool to elucidate the mechanisms of various biological processes related to vascular flow.
Topics: Animals; Cell Culture Techniques; Cells, Cultured; Fibroblasts; Human Umbilical Vein Endothelial Cells; Humans; Mice; Perfusion; Tissue Engineering
PubMed: 33112918
DOI: 10.1371/journal.pone.0240552 -
Nanomaterials (Basel, Switzerland) Jan 2021In the field of in vitro liver disease models, decellularised organ scaffolds maintain the original biomechanical and biological properties of the extracellular matrix...
In the field of in vitro liver disease models, decellularised organ scaffolds maintain the original biomechanical and biological properties of the extracellular matrix and are established supports for in vitro cell culture. However, tissue engineering approaches based on whole organ decellularized scaffolds are hampered by the scarcity of appropriate bioreactors that provide controlled 3D culture conditions. Novel specific bioreactors are needed to support long-term culture of bioengineered constructs allowing non-invasive longitudinal monitoring. Here, we designed and validated a specific bioreactor for long-term 3D culture of whole liver constructs. Whole liver scaffolds were generated by perfusion decellularisation of rat livers. Scaffolds were seeded with Luc+HepG2 and primary human hepatocytes and cultured in static or dynamic conditions using the custom-made bioreactor. The bioreactor included a syringe pump, for continuous unidirectional flow, and a circuit built to allow non-invasive monitoring of culture parameters and media sampling. The bioreactor allowed non-invasive analysis of cell viability, distribution, and function of Luc+HepG2-bioengineered livers cultured for up to 11 days. Constructs cultured in dynamic conditions in the bioreactor showed significantly higher cell viability, measured with bioluminescence, distribution, and functionality (determined by albumin production and expression of CYP enzymes) in comparison to static culture conditions. Finally, our bioreactor supports primary human hepatocyte viability and function for up to 30 days, when seeded in the whole liver scaffolds. Overall, our novel bioreactor is capable of supporting cell survival and metabolism and is suitable for liver tissue engineering for the development of 3D liver disease models.
PubMed: 33494337
DOI: 10.3390/nano11020275 -
Bioengineering (Basel, Switzerland) Aug 2021Drug and chemical development along with safety tests rely on the use of numerous clinical models. This is a lengthy process where animal testing is used as a standard... (Review)
Review
Drug and chemical development along with safety tests rely on the use of numerous clinical models. This is a lengthy process where animal testing is used as a standard for pre-clinical trials. However, these models often fail to represent human physiopathology. This may lead to poor correlation with results from later human clinical trials. Organ-on-a-Chip (OOAC) systems are engineered microfluidic systems, which recapitulate the physiochemical environment of a specific organ by emulating the perfusion and shear stress cellular tissue undergoes in vivo and could replace current animal models. The success of culturing cells and cell-derived tissues within these systems is dependent on the scaffold chosen; hence, scaffolds are critical for the success of OOACs in research. A literature review was conducted looking at current OOAC systems to assess the advantages and disadvantages of different materials and manufacturing techniques used for scaffold production; and the alternatives that could be tailored from the macro tissue engineering research field.
PubMed: 34436116
DOI: 10.3390/bioengineering8080113 -
Bioengineering & Translational Medicine Jan 2023Bioengineered tissues or organs produced using matrix proteins or components derived from xenogeneic sources pose risks of allergic responses, immune rejection, or even...
Bioengineered tissues or organs produced using matrix proteins or components derived from xenogeneic sources pose risks of allergic responses, immune rejection, or even autoimmunity. Here, we report successful xeno-free isolation, expansion, and cryopreservation of human endothelial cells (EC), fibroblasts (FBs), pericytes (PCs), and keratinocytes (KCs). We further demonstrate the bioprinting of a human skin substitute with a dermal layer containing xeno-free cultured human EC, FBs, and PCs in a xeno-free bioink containing human collagen type I and fibronectin layered in a biocompatible polyglycolic acid mesh and subsequently seeded with xeno-free human KCs to form an epidermal layer. Following implantation of such bilayered skin grafts on the dorsum of immunodeficient mice, KCs form a mature stratified epidermis with rete ridge-like structures. The ECs and PCs form human EC-lined perfused microvessels within 2 weeks after implantation, preventing graft necrosis, and eliciting further perfusion of the graft by angiogenic host microvessels. As proof-of-concept, we generated 12 individual grafts using a single donor of all four cell types. In summary, we describe the fabrication of a bioprinted vascularized bilayered skin substitute under completely xeno-free culture conditions demonstrating feasibility of a xeno-free approach to complex tissue engineering.
PubMed: 36684084
DOI: 10.1002/btm2.10324 -
Experimental Eye Research Jan 2022The human anterior segment perfusion culture model is a valuable tool for studying the trabecular meshwork (TM) and aqueous humor outflow in glaucoma. The traditional...
The human anterior segment perfusion culture model is a valuable tool for studying the trabecular meshwork (TM) and aqueous humor outflow in glaucoma. The traditional model relies on whole eye globes resulting in high cost and limited availability. Here, we developed a glue-based method which enabled us to use human corneal rims for perfusion culture experiments. Human corneal rim perfusion culture plates were 3D printed. Human corneal rims containing intact TM were attached and sealed to the plate using low viscosity and high viscosity glues, respectively. The human corneal rims were perfused using the constant flow mode, and the pressure changes were recorded using a computerized system. Outflow facility, TM stiffness, and TM morphology were evaluated. When perfused at rates from 1.2 to 3.6 μl/min, the outflow facility was 0.359 ± 0.216 μl/min/mmHg among 10 human corneal rims. The stiffness of the TM in naïve human corneal rim was similar to that of perfusion cultured human corneal rim. Also, the stiffness of TM of corneal rims perfused with dexamethasone was significantly higher than the control. Human corneal rims with glue contamination in the TM could be differentiated by high baseline intraocular pressure as well as high TM stiffness. Histology studies showed that the TM tissues perfused with plain medium appeared normal. We believed that our glued-based method is a useful tool and low-cost alternative to the traditional anterior segment perfusion culture model.
Topics: Aqueous Humor; Cornea; Elastic Modulus; Humans; Intraocular Pressure; Microscopy, Atomic Force; Models, Biological; Organ Culture Techniques; Tissue Adhesives; Tissue Donors; Trabecular Meshwork
PubMed: 34896309
DOI: 10.1016/j.exer.2021.108891 -
International Journal of Molecular... Sep 2021Drug-induced liver injury (DILI) is the major reason for failures in drug development and withdrawal of approved drugs from the market. Two-dimensional cultures of... (Review)
Review
Drug-induced liver injury (DILI) is the major reason for failures in drug development and withdrawal of approved drugs from the market. Two-dimensional cultures of hepatocytes often fail to reliably predict DILI: hepatoma cell lines such as HepG2 do not reflect important primary-like hepatic properties and primary human hepatocytes (pHHs) dedifferentiate quickly in vitro and are, therefore, not suitable for long-term toxicity studies. More predictive liver in vitro models are urgently required in drug development and compound safety evaluation. This review discusses available human hepatic cell types for in vitro toxicology analysis and their usage in established and emerging three-dimensional (3D) culture systems. Generally, 3D cultures maintain or improve primary hepatic functions (including expression of drug-metabolizing enzymes) of different liver cells for several weeks of culture, thus allowing long-term and repeated-dose toxicity studies. Spheroid cultures of pHHs have been comprehensively tested, but also other cell types such as HepaRG benefit from 3D culture systems. Emerging 3D culture techniques include usage of induced pluripotent stem-cell-derived hepatocytes and primary-like upcyte cells, as well as advanced culture techniques such as microfluidic liver-on-a-chip models. In-depth characterization of existing and emerging 3D hepatocyte technologies is indispensable for successful implementation of such systems in toxicological analysis.
Topics: Bioreactors; Cell Culture Techniques; Hepatocytes; Humans; Primary Cell Culture; Spheroids, Cellular; Toxicity Tests; Toxicology
PubMed: 34638555
DOI: 10.3390/ijms221910214 -
Scientific Reports Jun 2023Although a rare disease, rhabdomyosarcoma (RMS) is one of the most common cancers in children the more aggressive and metastatic subtype is the alveolar RMS (ARMS)....
Although a rare disease, rhabdomyosarcoma (RMS) is one of the most common cancers in children the more aggressive and metastatic subtype is the alveolar RMS (ARMS). Survival outcomes with metastatic disease remain dismal and the need for new models that recapitulate key pathological features, including cell-extracellular matrix (ECM) interactions, is warranted. Here, we report an organotypic model that captures cellular and molecular determinants of invasive ARMS. We cultured the ARMS cell line RH30 on a collagen sponge in a perfusion-based bioreactor (U-CUP), obtaining after 7 days a 3D construct with homogeneous cell distribution. Compared to static culture, perfusion flow induced higher cell proliferation rates (20% vs. 5%), enhanced secretion of active MMP-2, and upregulation of the Rho pathway, associated with cancer cell dissemination. Consistently, the ECM genes LAMA1 and LAMA2, the antiapoptotic gene HSP90, identified in patient databases as hallmarks of invasive ARMS, were higher under perfusion flow at mRNA and protein level. Our advanced ARMS organotypic model mimics (1) the interactions cells-ECM, (2) the cell growth maintenance, and (3) the expression of proteins that characterize tumor expansion and aggressiveness. In the future, the perfusion-based model could be used with primary patient-derived cell subtypes to create a personalized ARMS chemotherapy screening system.
Topics: Child; Humans; Rhabdomyosarcoma, Alveolar; Rhabdomyosarcoma; Cell Line; Perfusion; Cell Culture Techniques, Three Dimensional; Cell Line, Tumor
PubMed: 37296184
DOI: 10.1038/s41598-023-36210-4 -
Scientific Reports May 2023The development of microfluidic culture technology facilitates the progress of study of cell and tissue biology. This technology expands the understanding of...
The development of microfluidic culture technology facilitates the progress of study of cell and tissue biology. This technology expands the understanding of pathological and physiological changes. A skin chip, as in vitro model, consisting of normal skin tissue with epidermis and dermis layer (full thickness) was developed. Polydimethylsiloxane microchannels with a fed-batched controlled perfusion feeding system were used to create a full-thick ex-vivo human skin on-chip model. The design of a novel skin-on-a-chip model was reported, in which the microchannel structures mimic the architecture of the realistic vascular network as nutrients transporter to the skin layers. Viabilities of full-thick skin samples cultured on the microbioreactor and traditional tissue culture plate revealed that a precise controlled condition provided by the microfluidic enhanced tissue viability at least for seven days. Several advantages in skin sample features under micro-scale-controlled conditions were found such as skin mechanical strength, water adsorption, skin morphology, gene expression, and biopsy longevity. This model can provide an in vitro environment for localizing drug delivery and transdermal drug diffusion studies. The skin on the chip can be a valuable in vitro model for representing the interaction between drugs and skin tissue and a realistic platform for evaluating skin reaction to pharmaceutical materials and cosmetic products.
Topics: Humans; Skin; Epidermis; Microfluidics; Perfusion; Lab-On-A-Chip Devices
PubMed: 37258538
DOI: 10.1038/s41598-023-34796-3 -
Bioengineering (Basel, Switzerland) Mar 2022Fed-batch process intensification with a significantly shorter culture duration or higher titer for monoclonal antibody (mAb) production by Chinese hamster ovary (CHO)...
Fed-batch process intensification with a significantly shorter culture duration or higher titer for monoclonal antibody (mAb) production by Chinese hamster ovary (CHO) cells can be achieved by implementing perfusion operation at the N-1 stage for biomanufacturing. N-1 perfusion seed with much higher final viable cell density (VCD) than a conventional N-1 batch seed can be used to significantly increase the inoculation VCD for the subsequent fed-batch production (referred as N stage), which results in a shorter cell growth phase, higher peak VCD, or higher titer. In this report, we incorporated a process analytical technology (PAT) tool into our N-1 perfusion platform, using an in-line capacitance probe to automatically adjust the perfusion rate based on real-time VCD measurements. The capacitance measurements correlated linearly with the offline VCD at all cell densities tested (i.e., up to 130 × 10 cells/mL). Online control of the perfusion rate via the cell-specific perfusion rate (CSPR) decreased media usage by approximately 25% when compared with a platform volume-specific perfusion rate approach and did not lead to any detrimental effects on cell growth. This PAT tool was applied to six mAbs, and a platform CSPR of 0.04 nL/cell/day was selected, which enabled rapid growth and maintenance of high viabilities for four of six cell lines. In addition, small-scale capacitance data were used in the scaling-up of N-1 perfusion processes in the pilot plant and in the GMP manufacturing suite. Implementing a platform approach based on capacitance measurements to control perfusion rates led to efficient process development of perfusion N-1 for supporting high-density CHO cell cultures for the fed-batch process intensification.
PubMed: 35447688
DOI: 10.3390/bioengineering9040128 -
Biotechnology and Bioengineering Mar 2020Natural tissues are incorporated with vasculature, which is further integrated with a cardiovascular system responsible for driving perfusion of nutrient-rich oxygenated...
Natural tissues are incorporated with vasculature, which is further integrated with a cardiovascular system responsible for driving perfusion of nutrient-rich oxygenated blood through the vasculature to support cell metabolism within most cell-dense tissues. Since scaffold-free biofabricated tissues being developed into clinical implants, research models, and pharmaceutical testing platforms should similarly exhibit perfused tissue-like structures, we generated a generalizable biofabrication method resulting in self-supporting perfused (SSuPer) tissue constructs incorporated with perfusible microchannels and integrated with the modular FABRICA perfusion bioreactor. As proof of concept, we perfused an MLO-A5 osteoblast-based SSuPer tissue in the FABRICA. Although our resulting SSuPer tissue replicated vascularization and perfusion observed in situ, supported its own weight, and stained positively for mineral using Von Kossa staining, our in vitro results indicated that computational fluid dynamics (CFD) should be used to drive future construct design and flow application before further tissue biofabrication and perfusion. We built a CFD model of the SSuPer tissue integrated in the FABRICA and analyzed flow characteristics (net force, pressure distribution, shear stress, and oxygen distribution) through five SSuPer tissue microchannel patterns in two flow directions and at increasing flow rates. Important flow parameters include flow direction, fully developed flow, and tissue microchannel diameters matched and aligned with bioreactor flow channels. We observed that the SSuPer tissue platform is capable of providing direct perfusion to tissue constructs and proper culture conditions (oxygenation, with controllable shear and flow rates), indicating that our approach can be used to biofabricate tissue representing primary tissues and that we can model the system in silico.
Topics: Animals; Bioprinting; Bioreactors; Cell Line; Computer Simulation; Equipment Design; Hydrodynamics; Mice; Models, Biological; Osteoblasts; Perfusion
PubMed: 31788785
DOI: 10.1002/bit.27238