Three-dimensional cell culture of human mesenchymal stem cells in nanofibrillar cellulose hydrogels

Human mesenchymal stem cells (MSCs) are the most intensely studied and clinically used adult stem cell type. Conventional long-term cultivation of MSCs as a monolayer is known to result in a reduction of their functionality and viability. In addition, large volumes of cell culture medium are required to obtain cell quantities needed for their clinical use. In this proof of concept study, we cultivated human MSCs within a 3D nanofibrillar cellulose (NFC) hydrogel. We show that NFC is biocompatible with human MSCs, and represents a feasible approach to upscaling of their culture

Scientific, sustainability and regulatory challenges of cultured meat

Producing meat without the drawbacks of conventional animal agriculture would greatly contribute to future food and nutrition security. This Review Article covers biological, technological, regulatory and consumer acceptance challenges in this developing field of biotechnology. Cellular agriculture is an emerging branch of biotechnology that aims to address issues associated with the environmental impact, animal welfare and sustainability challenges of conventional animal farming for meat production. Cultured meat can be produced by applying current cell culture practices and biomanufacturing methods and utilizing mammalian cell lines and cell and gene therapy products to generate tissue or nutritional proteins for human consumption. However, significant improvements and modifications are needed for the process to be cost efficient and robust enough to be brought to production at scale for food supply. Here, we review the scientific and social challenges in transforming cultured meat into a viable commercial option, covering aspects from cell selection and medium optimization to biomaterials, tissue engineering, regulation and consumer acceptance

Plant Derived Cellulose Scaffolds as a Novel Biomaterial for 3D Cell Culture and Tissue Regeneration

This work presents an alternative approach to the production of cellulose-based biomaterials. Instead of extracting, processing and regenerating plant and or bacteria-derived cellulose into a biomaterial, my work established a decellularization protocol to remove cellular plant content from plant tissue resulting in a scaffold composed of cellulose with the evolved architecture of the plant cell wall. Tracheophyte plants, including clubmosses, horsetails, and ferns, gymnosperms and angiosperms, have evolved distinct vascular structures that support the transport of water and nutrients in xylem and phloem that form the vascular bundles (VBs)1. This thesis took it’s inspiration from the dense, linearly arranged, parallel microchannels which include (VBs) in the stalks of Asparagus officinalis possess an architecture with striking similarities to biomaterial scaffolds intended to repair damaged tissue. My work demonstrated that the plant cell wall contains many of the ideal characteristics of a medical biomaterial. The scaffold is biocompatible with mammalian cells and maintains high viability even with cell densities comparable to commercially available scaffolds. The cellulose scaffold could be biochemically functionalized or cross-linked to control the scaffolds' surface biochemistry and mechanical properties. My in vivo model demonstrated that the lignocellulose scaffold did not elicit a foreign body response. The scaffold was permissive to host cell invasion, including active host fibroblast, leading to the deposition of host collagen extracellular matrix. Importantly, active blood vessels formed within the scaffold to support the population of host cells. The scaffold retained much of its original shape and provided an inert, pro-vascular long-term environment for host cells to invade. Taken together, this led to the hypothesis that the innate plant cell wall architecture could restore the function of injured tissue, specifically that the vascular bundles could be used to promote axonal regeneration in spinal cord injuries. Rats with complete spinal cord transection were implanted with cellulose scaffolds with vascular bundles. Animals that received plant-derived scaffolds demonstrated a significant improvement in motor function. This thesis defines a novel and parallel route for exploiting naturally occurring plant microarchitectures of the underlying crystalline cellulose scaffold

Biocompatibility of Subcutaneously Implanted Plant-Derived Cellulose Biomaterials

ABSTRACTThere is intense interest in developing novel biomaterials which support the invasion and proliferation of living cells for potential applications in tissue engineering and regenerative medicine. Decellularization of existing tissues have formed the basis of one major approach to producing 3D scaffolds for such purposes. In this study, we utilize the native hypanthium tissue of apples and a simple preparation methodology to create implantable cellulose scaffolds. To examine biocompatibility, scaffolds were subcutaneously implanted in wild-type, immunocompetent mice (males and females; 6-9 weeks old). Following the implantation, the scaffolds were resected at 1, 4 and 8 weeks and processed for histological analysis (H&amp;E, Masson’s Trichrome, anti-CD31 and anti-CD45 antibodies). Histological analysis revealed a characteristic foreign body response to the scaffold 1 week post-implantation. However, the immune response was observed to gradually disappear by 8 weeks post-implantation. By 8 weeks, there was no immune response in the surrounding dermis tissue and active fibroblast migration within the cellulose scaffold was observed. This was concomitant with the deposition of a new collagen extracellular matrix. Furthermore, active blood vessel formation within the scaffold was observed throughout the period of study indicating the pro-angiogenic properties of the native scaffolds. Finally, while the scaffolds retain much of their original shape they do undergo a slow deformation over the 8-week length of the study. Taken together, our results demonstrate that native cellulose scaffolds are biocompatible and exhibit promising potential as a surgical biomaterial.</jats:p

Biocompatibility of Subcutaneously Implanted Plant-Derived Cellulose Biomaterials.

There is intense interest in developing novel biomaterials which support the invasion and proliferation of living cells for potential applications in tissue engineering and regenerative medicine. Decellularization of existing tissues have formed the basis of one major approach to producing 3D scaffolds for such purposes. In this study, we utilize the native hypanthium tissue of apples and a simple preparation methodology to create implantable cellulose scaffolds. To examine biocompatibility, scaffolds were subcutaneously implanted in wild-type, immunocompetent mice (males and females; 6-9 weeks old). Following the implantation, the scaffolds were resected at 1, 4 and 8 weeks and processed for histological analysis (H&E, Masson's Trichrome, anti-CD31 and anti-CD45 antibodies). Histological analysis revealed a characteristic foreign body response to the scaffold 1 week post-implantation. However, the immune response was observed to gradually disappear by 8 weeks post-implantation. By 8 weeks, there was no immune response in the surrounding dermis tissue and active fibroblast migration within the cellulose scaffold was observed. This was concomitant with the deposition of a new collagen extracellular matrix. Furthermore, active blood vessel formation within the scaffold was observed throughout the period of study indicating the pro-angiogenic properties of the native scaffolds. Finally, while the scaffolds retain much of their original shape they do undergo a slow deformation over the 8-week length of the study. Taken together, our results demonstrate that native cellulose scaffolds are biocompatible and exhibit promising potential as a surgical biomaterial

Cellulose scaffolds implantation and resection.

The subcutaneous implantations of cellulose scaffolds biomaterial were performed on the dorsal region of a C57BL/10ScSnJ mouse model by small skin incisions (8 mm) (A). Each implant was measured before their implantation for scaffold area comparison (B). Cellulose scaffolds were resected at 1 week (D), 4 weeks (E) and 8 weeks (F) after the surgeries and macroscopic pictures were taken (control skin in C). The changes in cellulose scaffold surface area over time are presented (G). The pre-implantation scaffold had an area of 26.30±1.98mm2. Following the implantation, the area of the scaffold declined to 20.74±1.80mm2 after 1 week, 16.41±2.44mm2 after 4 weeks and 13.82±3.88mm2 after 8 weeks. The surface area of the cellulose scaffold has a significant decrease of about 12mm2 (48%) after 8 weeks implantation (* = P<0.001; n = 12–14).</p

Cellulose scaffold preparation.

<p>Macroscopic appearance of a freshly cut apple hypanthium tissue (A) and the translucent cellulose scaffold biomaterial post-decellularization and absent of all native apple cells or cell debris (B). H&E staining of cross sectioned decellularized cellulose scaffold (<b>C</b>). The cell walls thickness and the absence of native apple cells following decellularization are shown. The 3D acellular and highly porous cellulose scaffold architecture is clearly revealed by scanning electron microscopy (D). Scale bar: A-B = 2mm, C-D = 100μm.</p

Cellulose scaffold preparation.

Macroscopic appearance of a freshly cut apple hypanthium tissue (A) and the translucent cellulose scaffold biomaterial post-decellularization and absent of all native apple cells or cell debris (B). H&E staining of cross sectioned decellularized cellulose scaffold (C). The cell walls thickness and the absence of native apple cells following decellularization are shown. The 3D acellular and highly porous cellulose scaffold architecture is clearly revealed by scanning electron microscopy (D). Scale bar: A-B = 2mm, C-D = 100μm.</p

Biocompatibility and cell infiltration.

Cross sections of representative cellulose scaffolds stained with H&E and anti-CD45. These global view show the acute moderate-severe anticipated foreign body reaction at 1 week (A), the mild chronic immune and subsequent cleaning processes at 4 weeks (B) and finally, the cellulose scaffold assimilated into the native mouse tissue at 8 weeks (C). Higher magnification regions of interest (D-F), see inset (A-C), allow the observation of all the cell type population within biomaterial assimilation processes. At 1 week, we can observe populations of granulocytes, specifically; polymorphonuclear (PMN) and eosinophils that characterize the acute moderate to severe immune response, a normal reaction to implantation procedures (D). At 4 weeks, a decreased immune response can be observed (mild to low immune response) and the population of cells within the epidermis surrounding scaffolds now contain higher levels of monocytes and lymphocytes characterizing chronic response (E). Finally, at 8 weeks, the immune response has completely resorbed with the epidermis tissue now appearing normal (F). The immune response observed with H&E staining is confirmed using anti-CD45 antibody, a well known markers of leukocytes (G-I). The population of cells within the scaffold are now mainly macrophages, multinucleated cells and active fibroblasts. Scale bars: A-C = 1mm, D-F = 100μm and G-I = 500μm.</p