Large-Scale Formation and Long-Term Culture of Hepatocyte Organoids From Streamlined In Vivo Genome-Edited GGTA1-/- Pigs for Bioartificial Liver Applications.

Hepatocyte transplantation and bioartificial liver (BAL) systems hold significant promise as less invasive alternatives to traditional transplantation, providing crucial temporary support for patients with acute and chronic liver failure. Although human hepatocytes are ideal, their use is limited by ethical concerns and donor availability, leading to the use of porcine hepatocytes in BAL systems due to their functional similarities. Recent advancements in gene-editing technology have improved porcine organ xenotransplantation clinical trials by addressing immune rejection issues. Gene-edited pigs, such as alpha-1,3-galactosyltransferase (GGTA1) knockout pigs, offer a secure source of primary cells for BAL systems. Our research focuses on optimizing the safety and functionality of porcine primary hepatocytes during large-scale cultivation. We achieved this by creating GGTA1 knockout pigs through one-step delivery of CRISPR/Cas9 to pig zygotes via oviduct injection of rAAV, and enhancing hepatocyte viability and function by co-culturing hepatocytes with Roof plate-specific spondin 1 overexpressing HUVECs (R-HUVECs). Using a Rocker culture system, approximately 1010 primary porcine hepatocytes and R-HUVECs rapidly formed organoids with a diameter of 92.1 ± 28.1 µm within 24 h. These organoids not only maintained excellent functionality but also supported partial hepatocyte self-renewal during long-term culture over 28 days. Gene-edited primary porcine hepatocyte organoids will significantly advance the applications of hepatocyte transplantation and BAL systems.

Visual screening of CRISPR/Cas9 editing efficiency based on micropattern arrays for editing porcine cells.

CRISPR/Cas9 technology, combined with somatic cell nuclear transplantation (SCNT), represents the primary approach to generating gene-edited pigs. The inefficiency in acquiring gene-edited nuclear donors is attributed to low editing and delivery efficiency, both closely linked to the selection of CRISPR/Cas9 forms. However, there is currently no direct method to evaluate the efficiency of CRISPR/Cas9 editing in porcine genomes. A platform based on fluorescence reporting signals and micropattern arrays was developed in this study, to visually assess the efficiency of gene editing. The optimal specifications for culturing porcine cells, determined by the quantity and state of cells grown on micropattern arrays, were a diameter of 200 µm and a spacing of 150 µm. By visualizing the area of fluorescence loss and measuring the gray value of the micropattern arrays, it was quickly determined that the mRNA form targeting porcine cells exhibited the highest editing efficiency compared to DNA and Ribonucleoprotein (RNP) forms of CRISPR/Cas9. Subsequently, four homozygotes of the ß4GalNT2 gene knockout were successfully obtained through the mRNA form, laying the groundwork for the subsequent generation of gene-edited pigs. This platform facilitates a quick, simple, and effective evaluation of gene knockout efficiency. Additionally, it holds significant potential for swiftly testing novel gene editing tools, assessing delivery methods, and tailoring evaluation platforms for various cell types.

One-step in vivo gene knock-out in porcine embryos using recombinant adeno-associated viruses.

Introduction: Gene-edited pigs have become prominent models for studying human disease mechanisms, gene therapy, and xenotransplantation. CRISPR (clustered regularly interspaced short palindromic repeats)/CRISPR-associated 9 (CRISPR/Cas9) technology is a widely employed tool for generating gene-edited pigs. Nevertheless, delivering CRISPR/Cas9 to pre-implantation embryos has traditionally posed challenges due to its reliance on intricate micromanipulation equipment and specialized techniques, resulting in high costs and time-consuming procedures. This study aims to introduce a novel one-step approach for generating genetically modified pigs by transducing CRISPR/Cas9 components into pre-implantation porcine embryos through oviductal injection of recombinant adeno-associated viruses (rAAV). Methods: We first used rAAV-1, rAAV-6, rAAV-8, rAAV-9 expressing EGFP to screen for rAAV serotypes that efficiently target porcine embryos, and then, to achieve efficient expression of CRISPR/Cas9 in vivo for a short period, we packaged sgRNAs targeting the GHR genes to self-complementary adeno-associated virus (scAAV), and Cas9 proteins to single-stranded adeno-associated virus (ssAAV). The efficiency of porcine embryos -based editing was then validated in vitro. The feasibility of this one-step method to produce gene-edited pigs using rAAV-CRISPR/Cas9 oviductal injection into sows within 24 h of conception was then validated. Results: Our research firstly establishes the efficient delivery of CRISPR/Cas9 to pig zygotes, both in vivo and in vitro, using rAAV6. Successful gene editing in pigs was achieved through oviductal injection of rAAV-CRISPR/Cas9. Conclusion: This method circumvents the intricate procedures involved in in vitro embryo manipulation and embryo transfers, providing a straightforward and cost-effective approach for the production of gene-edited pigs.

Autoclaving pHEMA-Based Hydrogels Immersed in Deionized Water has No Effect on Physicochemical Properties and Cell Behaviors.

Hydrogels based on poly-(2-hydroxyethyl methacrylate) (pHEMA) have been widely used as biomaterials in tissue engineering due to their biocompatibility, hydrophilicity, and low friction coefficient. The terminal sterilization of hydrogels is a critical step in clinical applications. However, regulations and standardization for the sterilization of hydrogels based on pHEMA are still lacking. In this study, we explored six sterilization methods on pHEMA-based materials (A1: pHEMA, A2: pHEMA copolymerizes with acrylic acid, and A3: pHEMA copolymerizes with acrylic acid and further coordinated with iron ions), such as gamma irradiation, 75% ethanol, ultraviolet (UV), ethylene oxide (EtO), and autoclaving with or without deionized water (autoclaving-H2O or autoclaving-dry). Combining results from the multifaceted approaches with assessment, pHEMA-based hydrogels can be completely sterilized via the autoclaving-H2O method analyzed by sterilized testing. The physicochemical properties and cell behavior of sterilized hydrogels were not influenced by this sterilization approach, validated by Fourier transform infrared (FT-IR) spectroscopy and tensile tests. The pHEMA-based hydrogel sterilized by the autoclaving-H2O method also had no effect on the cell behavior evaluated by in vitro cytotoxicity experiments and caused no evident inflammatory reaction in tissue in vivo implantation experiments. However, it was also found that there were still some defects in the A2 and A3 groups as biomaterials possibly because of an inappropriate proportion of formulations or raw material used in exploring sterilization methods. These findings have implications for the improvement and clinical application of pHEMA-based hydrogels.

Kidney ECM Pregel Nanoarchitectonics for Microarrays to Accelerate Harvesting Gene-Edited Porcine Primary Monoclonal Spheres.

One of the key steps of using CRISPR/Cas9 to obtain gene-edited cells used in generating gene-edited animals combined with somatic cell nuclear transplantation (SCNT) is to harvest monoclonal cells with genetic modifications. However, primary cells used as nuclear donors always grow slowly and fragile after a series of gene-editing operations. The extracellular matrix (ECM) formulated directly from different organs comprises complex proteins and growth factors that can improve and regulate the cellular functions of primary cells. Herein, sodium lauryl ether sulfate (SLES) detergent was first used to perfuse porcine kidney ECM, and the biological properties of the kidney ECM were optimized. Then, we used a porcine kidney ECM pregel to pattern the microarray and developed a novel strategy to shorten the time of obtaining gene-edited monoclonal cell spheroids with low damage in batches. Our results showed that the SLES-perfused porcine kidney ECM pregel displayed superior biological activities in releasing growth factors and promoting cell proliferation. Finally, combined with microarray technology, we quickly obtained monoclonal cells in good condition, and the cells used as nuclear donors to construct recombinant embryos showed a significantly higher success rate than those of the traditional method. We further successfully produced genetically edited pigs.

Ordered micropattern arrays fabricated by lung-derived dECM hydrogels for chemotherapeutic drug screening.

Aims: This study aims to evaluate ECM-coated micropattern arrays derived from decellularization of native porcine lungs as a novel three-dimensional cell culture platform. Methods: ECM derived from decellularization of native porcine lungs was exploited to prepare hydrogels. Then, dECM-coated micropattern arrays were fabricated at four different diameters (50, 100, 150 and 200 â€‹µm) using polydimethylsiloxane (PDMS). Two lung cancer cell lines, A549 and H1299, were tested on a dECM-coated micropattern array as a novel culture platform for cell adhesion, distribution, proliferation, viability, phenotype expression, and drug screening to evaluate the cytotoxicity of paclitaxel, doxorubicin and cisplatin. Results: The ECM derived from decellularization of native porcine lungs supported cell adhesion, distribution, viability and proliferation better than collagen I and Matrigel as the coated matrix on the surface. Moreover, the optimal diameter of the micropattern arrays was 100-150 â€‹µm, as determined by measuring the morphology, viability, proliferation and phenotype of the cancer cell spheroids. Cell spheroids of A549 and H1299 on dECM-coated micropattern arrays showed chemoresistance to anticancer drugs compared to that of the monolayer. The different distributions of HIF-1α, MCL-1 (in the center) and Ki-67 and MRP2 (in the periphery) of the spheroids demonstrated the good establishment of basal-lateral polarity and explained the chemoresistance phenomenon of spheroids. Conclusions: This novel three-dimensional cell culture platform is stable and reliable for anticancer drug testing. Drug screening in dECM-coated micropattern arrays provides a powerful alternative to existing methods for drug testing and metabolic profiling in the drug discovery process.

Fabrication of Size-Controllable and Arrangement-Orderly HepG2 Spheroids for Drug Screening via Decellularized Liver Matrix-Derived Micropattern Array Chips.

Three-dimensional (3D) culture via micropattern arrays to generate cellular spheroids seems a promising in vitro biomimetic system for liver tissue engineering applications, such as drug screening. Recently, organ-derived decellularized extracellular matrix emerges as arguably the most biomimetic bioink. Herein, decellularized liver matrix (DLM)-derived micropattern array chips were developed to fabricate size-controllable and arrangement-orderly HepG2 spheroids for drug screening. The porcine DLM was obtained by the removal of cellular components and then ground into powder, followed by enzymolysis. DLM as a coating substrate was compared with collagen type I (Col I) and Matrigel in terms of biological performance for enhancing cell adhesion, proliferation, and functions. Subsequently, we used poly(dimethylsiloxane) (PDMS) to adsorb DLM as the bioink to fabricate micropattern array chips. The optimal shape and size of micropattern were determined by evaluating the morphology, viability, and functions of HepG2 3D cellular aggregates. In addition, drug-susceptibility testing (paclitaxel, doxorubicin HCl, and disulfiram) was performed on this novel platform. The DLM provided the tissue-specific microenvironment that provided suitable supports for HepG2 cells, compared to Col I and Matrigel. A circular micropattern with a diameter of 100 µm was the optimal processing parameter to rapidly fabricate large-scale, size-controllable, and arrangement-orderly HepG2 cellular aggregates with 3D spheroid's shape and high cell viability. Drug screening testing showed that the effect of a drug could be directly demonstrated on-chip by confocal microscopy measuring the viability of spheroids. We provide a novel platform for the large-scale generation of HepG2 spheroids with uniform size and arrangement, thus bringing convenience, reducing error, and increasing reproducibility for a rapid drug discovery by fluorescence quantitative analysis. This methodology may be possible to apply in advancing personalized medicine and drug discovery.

Measurement and Destruction of Porcine Endogenous Retrovirus in the Chinese Bama Minipig.

Porcine hepatocytes are widely used in bioartificial liver (BAL) systems for the treatment of liver failure, and Chinese Bama minipigs (BMPs) are extensively used for animal experiments in the field of medicine in China. The genome of porcine endogenous retroviruses (PERVs) has not yet been accurately quantified, posing a threat to their clinical application because they act as a source of cells. In this study, we used genome sequence data from BMPs to predict PERV copies and their distribution. We validated and quantified the identified PERV copies and subtypes across different BMP individuals and tissues using quantitative real-time polymerase chain reaction and droplet digital polymerase chain reaction, respectively, and found that the BMP genome contains only 11 to 21 PERV copies. Notably, they lack the C subtype, which is a relatively safe cell source. Moreover, we applied CRISPR/Cas9 technology to knock out the pol fragment of PERVs in primary renal fibroblasts (PRFs) from BMPs and obtain PERV-destructed cells. Overall, our results lay a foundation for obtaining PERV-destructed BMPs as a safe source of hepatocytes for BALs for future applications.