Application of human mesenchymal and pluripotent stem cell microcarrier cultures in cellular therapy: Achievements and future direction

AbstractMesenchymal stem cells (MSCs) have recently made significant progress with multiple clinical trials targeting modulation of immune responses, regeneration of bone, cartilage, myocardia, and diseases like Metachromatic leukodystrophy and Hurler syndrome. On the other hand, the use of human embryonic and induced pluripotent stem cells (hPSCs) in clinical trials is rather limited mainly due to safety issues. Only two clinical trials, retinal pigment epithelial transplantation and treatment of spinal cord injury were reported. Cell doses per treatment can range between 50,000 and 6billion cells. The current 2-dimensional tissue culture platform can be used when low cell doses are needed and it becomes impractical when doses above 50million are needed. This demand for future cell therapy has reinvigorated interests in the use of the microcarrier platform for generating stem cells in a scalable 3-dimensional manner.Microcarriers developed for culturing adherent cell lines in suspension have been used mainly in vaccine production and research purposes. Since MSCs grow as monolayers similar to conventional adherent cell lines, adapting MSCs to a microcarrier based expansion platform has been progressing rapidly. On the other hand, establishing a robust microcarrier platform for hPSCs is more challenging as these cells grow in multilayer colonies on extracellular matrices and are more susceptible to shear stress.This review describes properties of commercially available microcarriers developed for cultivation of anchorage dependent cells and present current achievements for expansion and differentiation of stem cells. Key issues such as microcarrier properties and coatings, cell seeding conditions, medium development and improved bioprocess parameters needed for optimal stem cell systems are discussed

Long-term microcarrier suspension cultures of human embryonic stem cells

AbstractThe conventional method of culturing human embryonic stem cells (hESC) is on two-dimensional (2D) surfaces, which is not amenable for scale up to therapeutic quantities in bioreactors. We have developed a facile and robust method for maintaining undifferentiated hESC in three-dimensional (3D) suspension cultures on matrigel-coated microcarriers achieving 2- to 4-fold higher cell densities than those in 2D colony cultures. Stable, continuous propagation of two hESC lines on microcarriers has been demonstrated in conditioned media for 6 months. Microcarrier cultures (MC) were also demonstrated in two serum-free defined media (StemPro and mTeSR1). MC achieved even higher cell concentrations in suspension spinner flasks, thus opening the prospect of propagation in controlled bioreactors

Isolation and Chimerization of a Highly Neutralizing Antibody Conferring Passive Protection against Lethal Bacillus anthracis Infection

Several studies have demonstrated that the passive transfer of protective antigen (PA)-neutralizing antibodies can protect animals against Bacillus anthracis infection. The standard protocol for the isolation of PA-neutralizing monoclonal antibodies is based upon a primary selection of the highest PA-binders by ELISA, and usually yields only few candidates antibodies. We demonstrated that by applying a PA-neutralization functionality-based screen as the primary criterion for positive clones, it was possible to isolate more than 100 PA-neutralizing antibodies, some of which exhibited no measurable anti-PA titers in ELISA. Among the large panel of neutralizing antibodies identified, mAb 29 demonstrated the most potent activity, and was therefore chimerized. The variable region genes of the mAb 29 were fused to human constant region genes, to form the chimeric 29 antibody (cAb 29). Guinea pigs were fully protected against infection by 40LD50 B. anthracis spores following two separate administrations with 10 mg/kg of cAb 29: the first administration was given before the challenge, and a second dose was administered on day 4 following exposure. Moreover, animals that survived the challenge and developed endogenous PA-neutralizing antibodies with neutralizing titers above 100 were fully protected against repeat challenges with 40LD50 of B. anthracis spores. The data presented here emphasize the importance of toxin neutralization-based screens for the efficient isolation of protective antibodies that were probably overlooked in the standard screening protocol. The protective activity of the chimeric cAb 29 demonstrated in this study suggest that it may serve as an effective immunotherapeutic agent against anthrax

Pushing the envelope in tissue engineering: Ex vivo production of thick vascularized cardiac extracellular matrix constructs

Functional vascularization is a prerequisite for cardiac tissue engineering of constructs with physiological thicknesses. We previously reported the successful preservation of main vascular conduits in isolated thick acellular porcine cardiac ventricular ECM (pcECM). We now unveil this scaffold's potential in supporting human cardiomyocytes and promoting new blood vessel development ex vivo, providing long-term cell support in the construct bulk. A custom-designed perfusion bioreactor was developed to remodel such vascularization ex vivo, demonstrating, for the first time, functional angiogenesis in vitro with various stages of vessel maturation supporting up to 1.7 mm thick constructs. A robust methodology was developed to assess the pcECM maximal cell capacity, which resembled the human heart cell density. Taken together these results demonstrate feasibility of producing physiological-like constructs such as the thick pcECM suggested here as a prospective treatment for end-stage heart failure. Methodologies reported herein may also benefit other tissues, offering a valuable in vitro setting for "thick-tissue" engineering strategies toward large animal in vivo studies.Israeli Science Foundation/1563/10Singapore National Research Foundatio

An intermittent rocking platform for integrated expansion and differentiation of human pluripotent stem cells to cardiomyocytes in suspended microcarrier cultures

The development of novel platforms for large scale production of human embryonic stem cells (hESC) derived cardiomyocytes (CM) becomes more crucial as the demand for CMs in preclinical trials, high throughput cardio toxicity assays and future regenerative therapeutics rises. To this end, we have designed a microcarrier (MC) suspension agitated platform that integrates pluripotent hESC expansion followed by CM differentiation in a continuous, homogenous process. Hydrodynamic shear stresses applied during the hESC expansion and CM differentiation steps drastically reduced the capability of the cells to differentiate into CMs. Applying vigorous stirring during pluripotent hESC expansion on Cytodex 1 MC in spinner cultures resulted in low CM yields in the following differentiation step (cardiac troponin-T (cTnT): 22.83 ± 2.56%; myosin heavy chain (MHC): 19.30 ± 5.31%). Whereas the lower shear experienced in side to side rocker (wave type) platform resulted in higher CM yields (cTNT: 47.50 ± 7.35%; MHC: 42.85 ± 2.64%). The efficiency of CM differentiation is also affected by the hydrodynamic shear stress applied during the first 3 days of the differentiation stage. Even low shear applied continuously by side to side rocker agitation resulted in very low CM differentiation efficiency (cTnT < 5%; MHC < 2%). Simply by applying intermittent agitation during these 3 days followed by continuous agitation for the subsequent 9 days, CM differentiation efficiency can be substantially increased (cTNT: 65.73 ± 10.73%; MHC: 59.73 ± 9.17%). These yields are 38.3% and 39.3% higher (for cTnT and MHC respectively) than static culture control. During the hESC expansion phase, cells grew on continuously agitated rocker platform as pluripotent cell/MC aggregates (166 ± 88 × 105 μm2) achieving a cell concentration of 3.74 ± 0.55 × 106 cells/mL (18.89 ± 2.82 fold expansion) in 7 days. These aggregates were further differentiated into CMs using a WNT modulation differentiation protocol for the subsequent 12 days on a rocking platform with an intermittent agitation regime during the first 3 days. Collectively, the integrated MC rocker platform produced 190.5 ± 58.8 × 106 CMs per run (31.75 ± 9.74 CM/hESC seeded). The robustness of the system was demonstrated by using 2 cells lines, hESC (HES-3) and human induced pluripotent stem cell (hiPSC) IMR-90. The CM/MC aggregates formed extensive sarcomeres that exhibited cross-striations confirming cardiac ontogeny. Functionality of the CMs was demonstrated by monitoring the effect of inotropic drug, Isoproterenol on beating frequency. In conclusion, we have developed a simple robust and scalable platform that integrates both hESC expansion and CM differentiation in one unit process which is capable of meeting the need for large amounts of CMs

Expansion of human embryonic stem cells on cellulose microcarriers

This unit describes the routine maintenance and expansion of undifferentiated human embryonic stem cells (hESC) on cellulose microcarriers. Conventionally, hESCs have been maintained on feeder cells or extracellular matrixcoated two-dimensional tissue culture plates. The expansion of hESC on a tissue culture platform is limited by the available surface area and the requirement of repetitive subculturing to reach the required cell yield. Here, we show that expansion of hESC can be carried out in a three-dimensional suspension culture using Matrigel-coated cellulose microcarriers. hESCs from a tissue culture plate can be seeded directly onto the microcarriers; hESC microcarrier culture is passaged and expanded by mechanical dissociation of the cells without enzyme. Expansion of the culture in a 100-ml spinner flask is also described. Long-term culture of hESC on the microcarriers maintains typical pluripotent markers (OCT-4, Tra-1-60, and SSEA-4) and stable karyotype. Spontaneous differentiations of microcarrier-maintained hESCs in vitro (embryoid body formation) and in vivo (teratoma formation in SCID mouse) have demonstrated formation of the three germ layers. These protocols can also be applied equally well to human induced pluripotent stem cells

Meticulous optimization of cardiomyocyte yields in a 3-stage continuous integrated agitation bioprocess

Human pluripotent stem cells (hPSCs) can be a renewable source for generating cardiomyocyte (CM) for treating myocardial infraction. In our previous publication, we described an integrated microcarrier-based wave reactor process for the expansion and differentiation of hPSCs to CMs on a rocker based platform. However, this platform is limited in terms of linear scalability and CMs purity. The present study describes ways to overcome these limitations by the use of a stirred scalable platform and incorporation of an additional lactate based purification step which increases CM purity.Efficient CM differentiation in stirred spinners was achieved by (1) Addition of ascorbic acid (AS) during the differentiation phase which resulted in an increase of 38.42% in CM yield (0.84 ± 0.03 × 106vs 1.17 ± 0.07 × 106 CM/mL for cultures without AS and with AS respectively) and (2) Change of agitation regime to a shorter static intervals one (from 66 min off/6 min on (66/6) to 8 min off/1 min on (8/1)) during the first 3 days of differentiation which resulted in 22% increase in CM yield (1.50 ± 0.10 × 106vs 1.23 ± 0.07 × 106 CM/mL). The combination of AS addition and change in agitation regime resulted in a production yield of 1.50 ± 0.10 × 106 CM/mL which is comparable to that achieved in the rocker platform as described before (1.61 ± 0.36 × 106 CM/mL).Increase in CM purity was achieved by changing of culture medium to RPMI1640 (without glucose) + 5 mM lactate +0.6 mM AS at day 10 of differentiation which resulted in 44.5% increase in CM purity at day 15. The increase in purity of CMs was due to the death of the non-CM cells (~76% of cell death). It is important to note that in the absence of glucose, lactate was consumed at a rate of 0.01 mmol/106 cells/h. Addition of glucose, even in small amounts, during the purification step prevents the process of CM purification, due to the growth of the non-CM cell population.In summary, hPSC (hESC-HES3 and hiPSC-IMR90) can be efficiently differentiated to CMs in a scalable spinner process which integrates 7 days of expansion (3.01 ± 0.51 × 106 to 3.50 ± 0.65 × 106 cells/mL) followed by 10 days of WNT modulated CM differentiation and 5 days of lactate based purification. CM yield of 1.38 ± 0.22 × 106 to 1.29 ± 0.42 × 106 CM/mL with 72.5 ± 8.35% to 83.12 ± 8.73% cardiac troponin-T positive cells were obtained from these cultures. Keywords: Human pluripotent stem cells, Microcarriers, Spinner platform, Cardiomyocytes differentiation, Lactate purification, Ascorbic aci