Spatiotemporal tracking of cells in tissue‐engineered cardiac organoids

Cardiac tissue engineering aims to create myocardial patches for repair of defective or damaged native heart muscle. The inclusion of non‐myocytes in engineered cardiac tissues has been shown to improve the properties of cardiac tissue compared to tissues engineered from enriched populations of myocytes alone. While attempts have been made to mix non‐myocytes (fibroblasts, endothelial cells) with cardiomyocytes, very little is understood about how the tissue properties are affected by varying the respective ratios of the three cell types and how these cells assemble into functional tissues with time. The goal of this study was to investigate the effects of modulating the ratios of the three cell types and to spatially and temporally track cardiac tri‐cultures of cells. Primary neonatal cardiac fibroblasts and D4T endothelial cells were incubated in 5 µM CellTracker^? green dye and CellTracker^? red dye, respectively, while neonatal cardiomyocytes were labelled with 20 µg/mL DAPI. The non‐myocytes were seeded either sequentially (pre‐culture) or simultaneously (tri‐culture) in Matrigel‐coated microchannels and allowed to form organoids, as in our previous studies. We also varied the seeding percentage of cardiomyocytes while keeping the total cell number constant in an attempt to improve the functional properties of the organoids. Organoids were imaged on days 1 and 4. Endothelial cells were seen to aggregate into clusters when simultaneously tri‐cultured with myocytes and fibroblasts, while pre‐cultures contained elongated cells. Functional properties of organoids were improved by increasing the seeding percentage of enriched cardiomyocytes from 40% to 80%. Copyright © 2009 John Wiley & Sons, Ltd.     

Specific complexes derived from extracellular matrix facilitate generation of structural and drug‐responsive human salivary gland microtissues through maintenance stem cell homeostasis

Three‐dimensional cultured salivary glands (SGs) microtissues hold great potentials for clinical research. However, most SGs microtissues still lack convincing structure and function due to poor supplementation of factors to maintain stem cell homeostasis. Extracellular matrix (ECM) plays a crucial role in regulating stem cell behavior. Thus, it is necessary to model stem cell microenvironment in vitro by supplementing culture medium with proteins derived from ECM. We prepared specific complexes from human SG ECM (s‐Ecx) and analyzed the components of the s‐Ecx. Human SG epithelial and mesenchymal cells were used to generate microtissues, and the optimum seeding cell number and ratio of two cell types were determined. Then, the s‐Ecx was introduced to the culture medium to assess its effect on stem cell behavior. Multiple specific factors were presented in s‐Ecx. s‐Ecx promoted maintenance of the stem cell and formation of specific structures resembling that of salivary glands and containing mucins, which suggested stem cell differentiation potential. Moreover, treatment of the microtissues with s‐Ecx increased their sensitivity to neurotransmitters. On the basis of the analysis of components, we believed that the presented growth factors are able to interact with stem cell they encountered in vivo, which promote the capacity to maintain stem cell homeostasis. This work provided foundations to study molecular mechanism of stem cell homeostasis in SGs and develop novel therapies for dry mouth through new drug discovery and disease modeling.     

In vivo cartilage repair using adipose‐derived stem cell‐loaded decellularized cartilage ECM scaffolds

We have previously reported a natural, human cartilage ECM (extracellular matrix)‐derived three‐dimensional (3D) porous acellular scaffold for in vivo cartilage tissue engineering in nude mice. However, the in vivo repair effects of this scaffold are still unknown. The aim of this study was to further explore the feasibility of application of cell‐loaded scaffolds, using autologous adipose‐derived stem cells (ADSCs), for cartilage defect repair in rabbits. A defect 4 mm in diameter was created on the patellar groove of the femur in both knees, and was repaired with the chondrogenically induced ADSC–scaffold constructs (group A) or the scaffold alone (group B); defects without treatment were used as controls (group C). The results showed that in group A all defects were fully filled with repair tissue and at 6 months post‐surgery most of the repair site was filled with hyaline cartilage. In contrast, in group B all defects were partially filled with repair tissue, but only half of the repair tissue was hyaline cartilage. Defects were only filled with fibrotic tissue in group C. Indeed, histological grading score analysis revealed that an average score in group A was higher than in groups B and C. GAG and type II collagen content and biomechanical property detection showed that the group A levels approached those of normal cartilage. In conclusion, ADSC‐loaded cartilage ECM scaffolds induced cartilage repair tissue comparable to native cartilage in terms of mechanical properties and biochemical components. Copyright © 2012 John Wiley & Sons, Ltd.     

Characterization of biomaterial‐free cell sheets cultured from human oral mucosal epithelial cells

The purpose of this study was to report the characteristics of biomaterial‐free sheets cultured from human oral mucosal epithelial cells without fibrin support, in vitro and after transplantation to limbal‐deficient models. Human oral mucosal epithelial cells and limbal epithelial cells were cultured for 2 weeks, and the colony‐forming efficiency (CFE) rates were compared. Markers of stem cells (p63), cell proliferation (Ki‐67) and epithelial differentiation (cytokeratin; K1, K3, K4, K13) were observed in colonies and in biomaterial‐free sheets. Biomaterial‐free sheets which had been detached with 1% dispase or biomaterial‐free sheets generated by fibrin support were transplanted to 12 limbal‐deficient rabbit models. In vitro cell viability, in vivo stability and cytokeratin characteristics of biomaterial‐free sheets were compared with those of sheets formed by fibrin‐coated culture 1 week after transplantation. Mean CFE rate was significantly higher in human oral mucosal epithelial cells (44.8%) than in human limbal epithelial cells(17.7%). K3 and K4 were well expressed in both colonies and sheets. Biomaterial‐free sheets had two to six layers of stratified cells and showed an average of 79.8% viable cells in the sheets after detachment. Cytokeratin expressions of biomaterial‐free sheets were comparable to those of sheets cultured by fibrin support, in limbal‐deficient models. Both p63 and Ki‐67 were well expressed in colonies, isolated sheets and sheets transplanted to limbal‐deficient models. Our results suggest that biomaterial‐free sheets cultured from human oral mucosal epithelial cells without fibrin support can be an alternative option for cell therapy in use for the treatment of limbal‐deficient diseases. Copyright © 2014 John Wiley & Sons, Ltd.     

Cell extracts from spleen and adipose tissues restore function to irradiation‐injured salivary glands

A cell extract from whole bone marrow (BM), which we named “BM Soup,” has the property to restore saliva secretion to irradiation (IR)‐injured salivary glands (SGs). However, BM cell harvesting remains an invasive procedure for the donor. The main objective of this study was to test the therapeutic effect of “Cell Soups” obtained from alternate tissues, such as adipose‐derived stromal cells (ADSCs) and spleen cells to repair SGs. BM Soup, Spleen Soup, ADSC Soup, or saline (vehicle control) was injected intravenously into mice with IR‐injured SGs (13Gy). Results demonstrated that all three cell soups restored 65–70% of saliva secretion, protected acinar cells, blood vessels, and parasympathetic nerves, and increased cell proliferation. Although protein array assays identified more angiogenesis‐related growth factors in ADSC Soup, the length of its therapeutic efficiency on saliva flow was less than that of the BM Soup and Spleen Soup. Another objective of this study was to compare “Fresh” versus “Cryopreserved (?80 °C)” BM Soup. It was found that the therapeutic effect of 12‐month “Cryopreserved BM Soup” was comparable to that of “Fresh BM Soup” on the functional restoration of IR‐injured SGs. In conclusion, both Spleen Soup and ADSC Soup can be used to mitigate IR‐damaged SGs.     

Cell culture of differentiated human salivary epithelial cells in a serum‐free and scalable suspension system: The salivary functional units model

Saliva aids in digestion, lubrication, and protection of the oral cavity against dental caries and oropharyngeal infections. Reduced salivary secretion, below an adequate level to sustain normal oral functions, is unfortunately experienced by head and neck cancer patients treated with radiotherapy and by patients with Sjögren's syndrome. No disease‐modifying therapies exist to date to address salivary gland hypofunction (xerostomia, dry mouth) because pharmacotherapies are limited by the need for residual secretory acinar cells, which are lost at the time of diagnosis, whereas novel platforms such as cell therapies are yet immature for clinical applications. Autologous salivary gland primary cells have clinical utility as personalized cell therapies, if they could be cultured to a therapeutically useful mass while maintaining their in vivo phenotype. Here, we devised a serum‐free scalable suspension culture system that grows partially digested human salivary tissue filtrates composing of acinar and ductal cells attached to their native extracellular matrix components while retaining their 3D in vivo spatial organization; we have coined these salivary spheroids as salivary functional units (SFU). The proposed SFU culture system was sub‐optimal, but we have found that the cells could still survive and grow into larger salivary spheroids through cell proliferation and aggregation for 5 to 10 days within the oxygen diffusion rates in vitro. In summary, by using a less disruptive cell isolation procedure as the starting point for primary cell culture of human salivary epithelial cells, we demonstrated that aggregates of cells remained proliferative and continued to express acinar and ductal cell‐specific markers.     

The role of human fibronectin‐ or placenta basement membrane extract‐based gels in favouring the formation of polarized salivary acinar‐like structures

Head and neck cancer patients treated with radiotherapy commonly experience hyposalivation and oral/tooth infections, leading to a reduced quality of life. Clinical management is currently unsatisfactory for dry mouth. Thus, there is a need for growing salivary fluid‐secreting (acinar) cells for these patients. However, functionally‐grown salivary acinar cells are cultured in Matrigel, a product that cannot be used clinically, owing to its source from a mouse sarcoma. Therefore, finding a gel suitable for clinical use and possessing properties similar to that of Matrigel would allow biopsied salivary cells to be expanded in vitro and transplanted into the mouths of xerostomic patients. This study tested gels made with human placenta basement membrane extract (BME) or fibronectin for the growth and differentiation of human salivary biopsies into acinar cells. We report here that, following expansion of primary human salivary gland epithelial cells (huSGs) in serum‐free medium, using these gels (made from human proteins) allowed morphological and functional differentiation of salivary ductal cells into acinar‐like cells. These (human) gels gave comparable results to Matrigel, such as differentiation into polarized acinar 3D units or monolayers with tight junction proteins (claudin‐1, ‐2, ‐3) and exhibiting adequate transepithelial electrical resistance, acinar proteins (AQP5, α‐amylase, mucin‐1, NKCC1) and acinar adhesion‐related cell markers (CD44, CD166). Ultrastructural, mRNA and protein analyses confirmed the formation of differentiated acinar polarized cells. The mitotic activity was highest with human placenta BME gel. This human culture model provided a reproducible approach to studying human salivary cell expansion and differentiation for tissue engineering. Copyright © 2016 John Wiley & Sons, Ltd.     

Adipose‐derived stem cells induce vascular tube formation of outgrowth endothelial cells in a fibrin matrix

Vascularization of engineered tissues is one of the current challenges in tissue engineering. Several strategies aim to generate a prevascularized scaffold which can be implanted at sites of injury or trauma. Endothelial cells derived from peripheral blood (outgrowth endothelial cells, OECs) display promising features for vascular tissue engineering, including their autologous nature, capacity for proliferation and ability to form mature vessels. In this study we investigated the ability of OECs to form vascular structures in co‐culture with adipose‐derived stem cells (ASCs) in a fibrin matrix. Using microcarrier beads coated with OECs, we showed ingrowth of endothelial cells in the fibrin scaffold. Furthermore, co‐cultures with ASCs induced vessel formation, as evidenced by immunostaining for CD31. The degradation of fibrin is at least in part mediated by expression of matrix metalloproteinase‐14. Moreover, we showed OEC/ASC‐induced vessel‐like structure formation even in the absence of microcarrier beads, where increasing amounts of ASCs resulted in a denser tubular network. Our data add new insights into co‐culture‐induced vessel formation of outgrowth endothelial cells within a fibrin matrix in an autologous system. Copyright © 2012 John Wiley & Sons, Ltd.     

Towards reconstruction of epithelialized cartilages from autologous adipose tissue‐derived stem cells

Deformities of the upper airways, including those of the nose and throat, are typically corrected by reconstructive surgery. The use of autologous somatic stem cells for repair of defects could improve quality and outcomes of such operations. The present study explored the ability of paediatric adipose‐derived stem cells (pADSCs), a readily available source of autologous stem cells, to generate a cartilage construct with a functional epithelium. Paediatric ADSCs seeded on the biodegradable nanocomposite polymer, polyhedral oligomeric silsesquioxane poly(?‐caprolactone‐urea) urethane (POSS‐PCL), proliferated and differentiated towards mesenchymal lineages. The ADSCs infiltrated three‐dimensional POSS‐PCL nanoscaffold and chondroid matrix was observed throughout chondrogenically induced samples. In ovo chorioallantoic membrane‐grafted ADSC‐nanoscaffold composites were enwrapped by host vessels indicating good compatibility in an in vivo system. Furthermore, pADSCs could be induced to transdifferentiate towards barrier‐forming epithelial‐like cells. By combining differentiation protocols, it was possible to generate epithelial cell lined chondrogenic micromasses from the same pADSC line. This proof‐of‐concept study appears to be the first to demonstrate that individual pADSC lines can differentiate towards two different germ lines and be successfully co‐cultured. This has important implications for bioengineering of paediatric airways and further confirms the plastic nature of ADSCs. Copyright © 2016 John Wiley & Sons, Ltd.     

Heparin cross‐linked collagen sponge scaffolds improve functional regeneration of rat tracheal epithelium

Tracheal epithelial cells maintain airway homeostasis by mediating mucociliary clearance. Following tracheal reconstruction, timely epithelial regeneration is required to prevent respiratory compromise and infectious diseases. To achieve rapid tracheal epithelial regeneration, a heparin cross‐linked collagen sponge containing fibroblast growth factor‐2 (FGF‐2) was prepared as a graft for tracheal reconstruction. The heparin cross‐linked sponge exhibited a high FGF‐2 retaining capacity, and tracheal epithelial and mesenchymal cells cultured in this sponge containing FGF‐2 showed high proliferative capacities. Subsequently, heparin‐free collagen sponge scaffolds (C/F scaffold) and collagen sponge scaffolds cross‐linked with 10 μg/ml heparin retained FGF‐2 (C/H10/F scaffold), and were transplanted into rats with tracheal defects. Invasion of both epithelial and non‐epithelial cells was greater in rats treated with the C/H10/F scaffold at 1 week post‐transplantation than in rats treated with the C/F scaffold. Moreover, at 2 weeks after transplantation, improved cilia formation was observed in the C/H10/F scaffold group, with higher motility and more potent posterior–anterior flow generation than in the C/F scaffold group. These results suggest that heparin improves functional regeneration of tracheal epithelium. Copyright © 2017 John Wiley & Sons, Ltd.     

Reconstruction of hepatic stellate cell‐incorporated liver capillary structures in small hepatocyte tri‐culture using microporous membranes

In liver sinusoids, hepatic stellate cells (HSCs) locate the outer surface of microvessels to form a functional unit with endothelia and hepatocytes. To reconstruct functional liver tissue in vitro, formation of the HSC‐incorporated sinusoidal structure is essential. We previously demonstrated capillary formation of endothelial cells (ECs) in tri‐culture, where a polyethylene terephthalate (PET) microporous membrane was intercalated between the ECs and hepatic organoids composed of small hepatocytes (SHs), i.e. hepatic progenitor cells, and HSCs. However, the high thickness and low porosity of the membranes limited heterotypic cell–cell interactions, which are essential to form HSC–EC hybrid structures. Here, we focused on the effective use of the thin and highly porous poly( d , l ‐lactide‐co‐glycolide) (PLGA) microporous membranes in SH–HSC–EC tri‐culture to reconstruct the HSC‐incorporated liver capillary structures in vitro. First, the formation of EC capillary‐like structures was induced on Matrigel‐coated PLGA microporous membranes. Next, the membranes were stacked on hepatic organoids composed of small SHs and HSCs. When the pore size and porosity of the membranes were optimized, HSCs selectively migrated to the EC capillary‐like structures. This process was mediated in part by platelet‐derived growth factor (PDGF) signalling. In addition, the HSCs were located along the outer surface of the EC capillary‐like structures with their long cytoplasmic processes. In the HSC‐incorporated capillary tissues, SHs acquired high levels of differentiated functions, compared to those without ECs. This model will provide a basis for the construction of functional, thick, vascularized liver tissues in vitro. Copyright © 2012 John Wiley & Sons, Ltd.     

Application of marrow mesenchymal stem cell‐derived extracellular matrix in peripheral nerve tissue engineering

To advance molecular and cellular therapy into the clinic for peripheral nerve injury, modification of neural scaffolds with the extracellular matrix (ECM) of peripheral nerves has been established as a promising alternative to direct inclusion of support cells and/or growth factors within a neural scaffold, while cell‐derived ECM proves to be superior to tissue‐derived ECM in the modification of neural scaffolds. Based on the fact that bone marrow mesenchymal stem cells (BMSCs), just like Schwann cells, are adopted as support cells within a neural scaffold, in this study we used BMSCs as parent cells to generate ECM for application in peripheral nerve tissue engineering. A chitosan nerve guidance conduit (NGC) and silk fibroin filamentous fillers were respectively prepared for co‐culture with purified BMSCs, followed by decellularization to stimulate ECM deposition. The ECM‐modified NGC and lumen fillers were then assembled into a chitosan–silk fibroin‐based, BMSC‐derived, ECM‐modified neural scaffold, which was implanted into rats to bridge a 10 mm‐long sciatic nerve gap. Histological and functional assessments after implantation showed that regenerative outcomes achieved by our engineered neural scaffold were better than those achieved by a plain chitosan–silk fibroin scaffold, and suggested the benefits of BMSC‐derived ECM for peripheral nerve repair. Copyright © 2016 John Wiley & Sons, Ltd.     

Up-regulated PAR-2-mediated salivary secretion in mice deficient in muscarinic acetylcholine receptor subtypes

Protease-activated receptor-2 (PAR-2) is expressed in the salivary glands and is expected to be a new target for the treatment of exocrine dysfunctions, such as dry mouth; however, the salivary secretory mechanism mediated by PAR-2 remains to be elucidated. Therefore, mechanism of the PAR-2-mediated salivary secretion was investigated in this study. We found that a PAR-2 agonist peptide, SLIGRL-OH, induced salivary flow in vivo and dose-dependent increase in [Ca(2+)](i) submandibular gland (SMG) acinar cells in wild-type (WT) mice and mice lacking M(3) or both M(1) and M(3) muscarinic acetylcholine receptors (mAChRs), whereas secretions in PAR-2 knockout (PAR-2KO) mice were completely abolished. The saliva composition secreted by SLIGRL-OH was similar to that secreted by mAChR stimulation. Ca(2+) imaging in WT acinar cells and beta-galactosidase staining in PAR-2KO mice, in which the beta-galactosidase gene (LacZ) was incorporated into the disrupted gene, revealed a nonubiquitous, sporadic distribution of PAR-2 in the SMG. Furthermore, compared with the secretion in WT mice, PAR-2-mediated salivary secretion and Ca(2+) response were enhanced in mice lacking M(3) or both M(1) and M(3) mAChRs, in which mAChR-stimulated secretion and Ca(2+) response in acinar cells were severely impaired. Although the mechanism underlying the enhanced PAR-2-mediated salivary secretion in M(3)-deficient mice is not clear, the result suggests the presence of some compensatory mechanism involving PAR-2 in the salivary glands deficient in cholinergic activation. These results indicate that PAR-2 present in the salivary glands mediates Ca(2+)-dependent fluid secretion, demonstrating potential usefulness of PAR-2 as a target for dry mouth treatment.     

Design and validation of a dynamic cell‐culture system for bone biology research and exogenous tissue‐engineering applications

Bone lacunocanalicular fluid flow ensures chemotransportation and provides a mechanical stimulus to cells. Traditional static cell‐culture methods are ill‐suited to study the intricacies of bone biology because they ignore the three‐dimensionality of meaningful cellular networks and the lacunocanalicular system; furthermore, reliance on diffusion alone for nutrient supply and waste product removal effectively limits scaffolds to 2–3 mm thickness. In this project, a flow‐perfusion system was custom‐designed to overcome these limitations: eight adaptable chambers housed cylindrical cell‐seeded scaffolds measuring 12 or 24 mm in diameter and 1–10 mm in thickness. The porous scaffolds were manufactured using a three‐dimensional (3D) periodic microprinting process and were composed of hydroxyapatite/tricalcium phosphate with variable thicknesses, strut sizes, pore sizes and structural configurations. A multi‐channel peristaltic pump drew medium from parallel reservoirs and perfused it through each scaffold at a programmable rate. Hermetically sealed valves permitted sampling or replacement of medium. A gas‐permeable membrane allowed for gas exchange. Tubing was selected to withstand continuous perfusion for > 2 months without leakage. Computational modelling was performed to assess the adequacy of oxygen supply and the range of fluid shear stress in the bioreactor–scaffold system, using 12 × 6 mm scaffolds, and these models suggested scaffold design modifications that improved oxygen delivery while enhancing physiological shear stress. This system may prove useful in studying complex 3D bone biology and in developing strategies for engineering thick 3D bone constructs. Copyright © 2013 John Wiley & Sons, Ltd.     

Current therapies for xerostomia and salivary gland hypofunction associated with cancer therapies

In cancer patients, as in the general population, medication is the most common cause of xerostomia. In general, saliva flow in these patients can be stimulated by mechanical or pharmacological stimulation of the salivary glands. Painful damaged oral mucosa can be treated by softening, lubricating mouthwashes or gels. A specific group of patients are those receiving radiotherapy for malignant tumours in the head and neck region. This treatment is inevitably associated with damages to the oral tissues, including the salivary glands, resulting in salivary gland hypofunction. When residual secretory capacity is present, it is advisable to stimulate the salivary glands by mechanical or gustatory stimuli regularly in these patients as supportive oral care. Alternatively, salivary flow can be stimulated by the use of cholinergic pharmaceutical preparations, such as pilocarpine or cevimeline. After the radiation therapy is ended, a dental check-up should be done every 3 months to allow control of any incipient oral inflammation and dental decay. For daily use, a special dentifrice (e.g. children's toothpaste) is recommended, since the taste of a regular dentifrice may be too strong for these patients. Nocturnal oral dryness can be alleviated by spraying the oral surfaces with water, or by applying a small amount of dentifrice on the dental smooth surfaces. When stimulation of salivary secretion fails, patients can be given palliative oral care in the form of application of mouthwashes and saliva substitutes. The daily use of a mouthwash, e.g. Biotène, Oral Balance or Zendium, or one of the saliva substitutes is indicated. Different types of saliva substitutes are now commercially available, containing different polymers as thickening agents, e.g. carboxymethylcellulose (Oralube and Glandosane), polyacrylic acid, and xanthan gum (Xialine). Recent developments, which are, however, still in the experimental stage, are bio-active saliva substitutes and mouthwashes containing antimicrobial peptides to protect the oral tissues against microbial colonization and to suppress and to cure mucosal and gingival inflammation.     

A humanised tissue‐engineered bone model allows species‐specific breast cancer‐related bone metastasis in vivo

Bone metastases frequently occur in the advanced stages of breast cancer. At this stage, the disease is deemed incurable. To date, the mechanisms of breast cancer‐related metastasis to bone are poorly understood. This may be attributed to the lack of appropriate animal models to investigate the complex cancer cell–bone interactions. In this study, two established tissue‐engineered bone constructs (TEBCs) were applied to a breast cancer‐related metastasis model. A cylindrical medical‐grade polycaprolactone‐tricalcium phosphate scaffold produced by fused deposition modelling (scaffold 1) was compared with a tubular calcium phosphate‐coated polycaprolactone scaffold fabricated by solution electrospinning (scaffold 2) for their potential to generate ectopic humanised bone in NOD/SCID mice. While scaffold 1 was found not suitable to generate a sufficient amount of ectopic bone tissue due to poor ectopic integration, scaffold 2 showed excellent integration into the host tissue, leading to bone formation. To mimic breast cancer cell colonisation to the bone, MDA‐MB‐231, SUM1315, and MDA‐MB‐231BO breast cancer cells were cultured in polyethylene glycol‐based hydrogels and implanted adjacent to the TEBCs. Histological analysis indicated that the breast cancer cells induced an osteoclastic reaction in the TEBCs, demonstrating analogies to breast cancer‐related bone metastasis seen in patients.     

Osteoarthritic human chondrocytes proliferate in 3D co‐culture with mesenchymal stem cells in suspension bioreactors

Osteoarthritis (OA) is a painful disease, characterized by progressive surface erosion of articular cartilage. The use of human articular chondrocytes (hACs) sourced from OA patients has been proposed as a potential therapy for cartilage repair, but this approach is limited by the lack of scalable methods to produce clinically relevant quantities of cartilage‐generating cells. Previous studies in static culture have shown that hACs co‐cultured with human mesenchymal stem cells (hMSCs) as 3D pellets can upregulate proliferation and generate neocartilage with enhanced functional matrix formation relative to that produced from either cell type alone. However, because static culture flasks are not readily amenable to scale up, scalable suspension bioreactors were investigated to determine if they could support the co‐culture of hMSCs and OA hACs under serum‐free conditions to facilitate clinical translation of this approach. When hACs and hMSCs (1:3 ratio) were inoculated at 20,000 cells/ml into 125‐ml suspension bioreactors and fed weekly, they spontaneously formed 3D aggregates and proliferated, resulting in a 4.75‐fold increase over 16 days. Whereas the apparent growth rate was lower than that achieved during co‐culture as a 2D monolayer in static culture flasks, bioreactor co‐culture as 3D aggregates resulted in a significantly lower collagen I to II mRNA expression ratio and more than double the glycosaminoglycan/DNA content (5.8 vs. 2.5 μg/μg). The proliferation of hMSCs and hACs as 3D aggregates in serum‐free suspension culture demonstrates that scalable bioreactors represent an accessible platform capable of supporting the generation of clinical quantities of cells for use in cell‐based cartilage repair.     

Three‐dimensional culture of mouse bone marrow cells within a porous polymer scaffold: effects of oxygen concentration and stromal layer on expansion of haematopoietic progenitor cells

To establish an ex vivo expansion method of haematopoietic progenitor cells (HPCs) and erythroid cells, three‐dimensional (3D) cultures of mouse bone marrow cells were performed, employing a porous polyvinyl formal (PVF) resin as a scaffold. In these cultures, the effects of oxygen concentration and co‐cultures with stromal cells on the expansion of HPCs and erythroid cells were investigated. When bone marrow cells were cultured under 3D conditions, HPCs and erythroid cells expanded without supplementation of exogenous cytokines, irrespective of the presence of stromal cells. On the contrary, slight expansion of HPCs or erythroid cells was observed in monolayer cultures as controls, indicating that the 3D cultures using the PVF scaffold were far better in expanding HPCs and erythroid cells than the monolayer cultures. Under hypoxic conditions, bone marrow stromal cells allowed for a 3D culture of erythroid cells and HPCs at higher cell densities compared to cultures without stromal cells, and the duration of the expansion of HPCs and erythroid cells after initiating the 3D co‐cultures was prolonged. The number of these cells increased throughout the culture period up to 3 weeks under hypoxic conditions, although the number decreased after 2 weeks under normoxic conditions. In conclusion, the 3D co‐culture method of haematopoietic cells with stromal cells under hypoxic conditions was confirmed to be effective in expanding HPCs and erythroid cells, and this method seemed to be useful for developing an ex vivo expansion method for haematopoietic cells. Copyright © 2010 John Wiley & Sons, Ltd.