Arindom Sen

Identification of growth and attachment factors for the serum-free isolation and expansion of human mesenchymal stromal cells.

BACKGROUND AIMS Ex vivo propagation of sparse populations of human mesenchymal stromal cells (hMSC) is critical for generating numbers sufficient for therapeutic applications. hMSC culture media have typically been supplemented with animal serum and, recently, human-sourced materials. However, these supplements are ill-defined and, thus, undesirable for clinical and research applications. Previously reported efforts to develop defined media for hMSC culture only resulted in slow or limited proliferation, and were unsuccessful in expanding these cells from primary cultures. Therefore a major step forward would be the identification of defined, serum-free culture conditions capable of supporting both the isolation and rapid expansion of hMSC. METHODS Using classical approaches of medium development, we were able to identify a set of growth and attachment factors that allowed the serum-free isolation and expansion of hMSC from bone marrow. RESULTS Heparin, selenium and platelet-derived growth factor (PDGF)-BB were found to be inhibitory for the growth of hMSC, whereas basic fibroblast growth factor (bFGF) was critical and worked synergistically with transforming growth factor (TGF)-beta1 to allow significant cell expansion. Ascorbic acid, hydrocortisone and fetuin were also found to be important growth and attachment factors that, in conjunction with substrate-coating proteins, allowed the isolation of hMSC from primary culture and their subsequent expansion. CONCLUSIONS We report a defined medium formulation (PPRF-msc6), consisting of key recombinant and serum-derived components, for the rapid isolation and expansion of hMSC in the absence of serum. This work represents an important step forward for achieving an ideal, completely defined synthetic medium composition for the safe use of hMSC in clinical settings.

Expansion of mammalian neural stem cells in bioreactors: effect of power input and medium viscosity

Multipotent neural precursors can be cultured in suspension bioreactors as aggregates of stem cells and progenitor cells. However, it is important to limit the size of the aggregates, as necrotic centers may develop at very large diameters. Previously, we have shown that the hydrodynamics within a suspension bioreactor can be used to control the diameter of NSC aggregates (D(MAVG)<150 microm) below sizes where necrosis would be expected to occur. In the present study, power law correlations were developed for our bioreactors showing the dependence of the maximum mean aggregate diameter on both the kinematic viscosity of the medium and the power input per unit mass of medium. The power input was manipulated by changing the agitation rate (60-100 rpm), and the viscosity was manipulated through the addition of non-toxic levels of carboxymethylcellulose. The study also confirmed that the maximum liquid shear generated at the surface of the aggregates was sufficient to dislodge single cells, thus limiting the maximum diameter of the aggregates, without causing cell damage (tau(max)=9.76 dyn/cm(2)). This is a first step in the development of a reproducible, scaled-up process for the production of neural stem cells for therapeutic applications including the treatment of neurodegenerative disorders and acute central nervous system injuries.

Spinal GABAergic transplants attenuate mechanical allodynia in a rat model of neuropathic pain.

Injury to the spinal cord or peripheral nerves can lead to the development of allodynia due to the loss of inhibitory tone involved in spinal sensory function. The potential of intraspinal transplants of GABAergic cells to restore inhibitory tone and thus decrease pain behaviors in a rat model of neuropathic pain was investigated. Allodynia of the left hind paw was induced in rats by unilateral L5– 6 spinal nerve root ligation. Mechanical sensitivity was assessed using von Frey filaments. Postinjury, transgenic fetal green fluorescent protein mouse GABAergic cells or human neural precursor cells (HNPCs) expanded in suspension bioreactors and differentiated into a GABAergic phenotype were transplanted into the spinal cord. Control rats received undifferentiated HNPCs or cell suspension medium only. Animals that received either fetal mouse GABAergic cell or differentiated GABAergic HNPC intraspinal transplants demonstrated a significant increase in paw withdrawal thresholds at 1 week post‐transplantation that was sustained for 6 weeks. Transplanted fetal mouse GABAergic cells demonstrated immunoreactivity for glutamic acid decarboxylase and GABA that colocalized with green fluorescent protein. Intraspinally transplanted differentiated GABAergic HNPCs demonstrated immunoreactivity for GABA and β‐III tubulin. In contrast, intraspinal transplantation of undifferentiated HNPCs, which predominantly differentiated into astrocytes, or cell suspension medium did not affect any behavioral recovery. Intraspinally transplanted GABAergic cells can reduce allodynia in a rat model of neuropathic pain. In addition, HNPCs expanded in a standardized fashion in suspension bioreactors and differentiated into a GABAergic phenotype may be an alternative to fetal cells for cell‐based therapies to treat chronic pain syndromes.

Large-scale expansion of mammalian neural stem cells: a review.

A relatively new approach to the treatment of neurodegenerative diseases is the direct use of neural stem cells (NSCs) as therapeutic agents. The expected demand for treatment from the millions of afflicted individuals, coupled with the expected demand from biotechnology companies creating therapies, has fuelled the need to develop large-scale culture methods for these cells. The rapid pace of discovery in this area has been assisted through the use of animal model systems, enabling many experiments to be performed quickly and effectively. This review focuses on recent developments in expanding human and murine NSCs on a large scale, including the development of new serum-free media and bioreactor protocols. In particular, engineering studies that characterise important scale-up parameters are examined, including studies examining the effects of long-term culture of NSCs in suspension bioreactors. In addition, recent advances in the human NSC system are reviewed, including techniques for the evaluation of NSC characteristics.

Improved expansion of human bone marrow-derived mesenchymal stem cells in microcarrier-based suspension culture.

Human bone marrow‐derived mesenchymal stem cells (hBM‐MSCs) have potential clinical utility in the treatment of a multitude of ailments and diseases, due to their relative ease of isolation from patients and their capacity to form many cell types. However, hBM‐MSCs are sparse, and can only be isolated in very small quantities, thereby hindering the development of clinical therapies. The use of microcarrier‐based stirred suspension bioreactors to expand stem cell populations offers an approach to overcome this problem. Starting with standard culture protocols commonly reported in the literature, we have successfully developed new protocols that allow for improved expansion of hBM‐MSCs in stirred suspension bioreactors using CultiSpher‐S microcarriers. Cell attachment was facilitated by using intermittent bioreactor agitation, removing fetal bovine serum, modifying the stirring speed and manipulating the medium pH. By manipulating these parameters, we enhanced the cell attachment efficiency in the first 8 h post‐inoculation from 18% (standard protocol) to 72% (improved protocol). Following microcarrier attachment, agitation rate was found to impact cell growth kinetics, whereas feeding had no significant effect. By serially subculturing hBM‐MSCs using the new suspension bioreactor protocols, we managed to obtain cell fold increases of 103 within 30 days, which was superior to the 200‐fold increase obtained using the standard protocol. The cells were found to retain their defining characteristics after several passages in suspension. This new bioprocess represents a more efficient approach for generating large numbers of hBM‐MSCs in culture, which in turn should facilitate the development of new stem cell‐based therapies. Copyright

Scale-Up of Breast Cancer Stem Cell Aggregate Cultures to Suspension Bioreactors†

It has been hypothesized that breast tumor formation results from the activity of a scarce population of cells known as Breast Cancer Stem Cells (BrCSCs) and that the development of effective breast cancer therapies may therefore ultimately rely upon the ability to effectively target these cells for eradication. The scarcity of BrCSCs in vivo severely compromises research on these populations, as analyses are restricted to those requiring small cell numbers, and has become a major impediment to the development of therapeutic strategies against breast cancer. Through the culture of murine tissue aggregates containing a population of BrCSCs, this study demonstrates the ability of propagating this scarce population in a controlled and reproducible manner, within suspension bioreactors. A rigorous theoretical framework has been developed in order to understand and characterize the implications of oxygen mass transfer within aggregates upon scale‐up and thereby provide a foundation for the scale‐up of aggregate cultures. A two‐factor, two‐level factorial experimental design was also performed in order to assess the effects of inoculation density and hydrodynamic shear upon cell yield. We discovered that the culture of the murine aggregates in a relatively low shear environment (τmax = 0.20 Pa) and inoculated at 3.50 × 104 cells/mL resulted in the best yields for the range of conditions investigated in suspension bioreactors. A detailed study on the oxygen uptake kinetics of the aggregates also revealed that the uptake rates were not significantly affected by mass transfer limitations, as uptake rates of aggregate cultures were found to be comparable to those observed in single cell cultures. Cells propagated in a process controlled 500 mL suspension bioreactor resulted in growth kinetics that were comparable to those observed in 125 mL bioreactors. Doubling times in the 500 mL vessel were found to be 23.9 h and attained a maximum cell density of 1.20 × 106 cells/mL. After enumerating the number of BrCSCs, this resulted in an approximately 20‐fold increase in BrCSC numbers in batch suspension cultures. With greater attention being applied to BrCSCs, their propagation in suspension bioreactors makes available experimental avenues that are not currently accessible and may thereby enable the development of more effective therapeutic drugs for the treatment of breast cancer.

Passaging Protocols for Mammalian Neural Stem Cells in Suspension Bioreactors

Mammalian neural stem cells (NSC) offer great promise as therapeutic agents for the treatment of central nervous system disorders. As a consequence of the large numbers of cells that will be needed for drug testing and transplantation studies, it is necessary to develop protocols for the large‐scale expansion of mammalian NSC. Neural stem cells and early progenitor cells can be expanded in vitro as aggregates in controlled bioreactors using carefully designed media. The first objective of this study was to determine if it is possible to maintain a population of murine neural stem and progenitor cells as aggregates in suspension culture bioreactors over extended periods of time. We discovered that serial passaging of a mixture of aggregates sizes resulted in high viabilities, high viable cell densities, and good control of aggregate diameter. When the NSC aggregates were serially subcultured three times without mechanical dissociation, a total multiplication ratio of 2.9 × 103 was achieved over a period of 12 days, whereas the aggregate size was controlled (mean diameter less than 150 μm) below levels at which necrosis would occur. Moreover, cell densities of 1.0 × 106 cells/mL were repeatedly achieved in batch culture with viabilities exceeding 80%. The second objective was to examine the proliferative potential of single cells shed from the surface of these aggregates. We found that the single cells, when subcultured, retained the capacity to generate new aggregates, gave rise to cultures with high viable cell densities and were able to differentiate into all of the primary cell phenotypes in the central nervous system.

Expansion of Human Neural Precursor Cells in Large-Scale Bioreactors for the Treatment of Neurodegenerative Disorders

The transplantation of in vitro expanded human neural precursor cells (hNPCs) represents a potential new treatment alternative for individuals suffering from incurable neurodegenerative disorders such as Parkinsons disease (PD) and Huntingtons disease (HD). However, in order for cell restorative therapy to have widespread therapeutic significance, it will be necessary to generate unlimited quantities of clinical grade hNPCs in a standardized method. We report here that we have developed a serum‐free medium and scale‐up protocols that allow for the generation of clinical quantities of human telencephalon‐derived hNPCs in 500‐mL computer‐controlled suspension bioreactors. The average hNPC aggregate diameter in the bioreactors was maintained below a target value of 500 μm by controlling the liquid shear field. The human cells, which were inoculated at 105 cells/mL, exhibited a doubling time of 84 h, underwent a 36‐fold expansion over the course of 18 days, and maintained an average viability of over 90%. The bioreactor‐derived hNPCs retained their nestin expression following expansion and were able to differentiate into glial and neuronal phenotypes under defined conditions. It has also been demonstrated that these hNPCs differentiated to a GABAergic phenotype that has recently been shown to be able to restore functional behavior in rat models of HD and neuropathic pain (Mukhida, K. et al. Stem Cells 2007; DOI 10.1634/stemcells.2007–0326). This study demonstrates that clinical quantities of hNPCs can be successfully and reproducibly generated under standardized conditions in computer‐controlled suspension bioreactors.

Human mesenchymal stem cell culture: rapid and efficient isolation and expansion in a defined serum-free medium.

Human mesenchymal stem cells (hMSCs) are typically obtained for research or therapeutic applications by isolating and subculturing adherent cells from bone marrow on tissue‐culture substrates using growth media. The purity and properties of the resulting populations can be affected greatly by the conditions under which they are cultured. Fetal bovine serum (FBS), although ill‐defined, has been widely used as a critical requirement for conventional hMSC culture. However, a defined serum‐free medium would greatly facilitate the development of robust, clinically acceptable bioprocesses for reproducibly generating quality‐assured cells. The present study provides evidence demonstrating that a defined serum‐free medium (PPRF‐msc6) shows several beneficial features over a conventional FBS‐containing medium for the production of hMSCs. When compared to control FBS‐based cultures, PPRF‐msc6 medium supported the derivation of hMSCs from primary cultures of bone marrow cells in a more rapid and consistent manner. Furthermore, hMSCs cultured in PPRF‐msc6 exhibited: (a) a greater colony‐forming capacity in primary as well as passaged cultures; (b) negligible lag phase and explicit exponential growth; (c) lower population doubling times (21–26 h vs 35–38 h between passage levels 1 and 10); (d) a greater number of population doublings (62 ± 4 vs 43 ± 2; over a 2 month period); and (e) a higher degree of homogeneity in size. Our data demonstrate that PPRF‐msc6 is an important development which opens the door for the rapid, efficient and reproducible production of hMSCs in clinical settings. Copyright

New Tissue Dissociation Protocol for Scaled-up Production of Neural Stem Cells in Suspension Bioreactors

The successful dissociation of mammalian neural stem cell (NSC) aggregates (neurospheres) into a single-cell suspension is an important procedure when expanding NSCs for clinical use, or when performing important assays such as clonal analyses. Until now, researchers have had to rely primarily on destructive mechanical methods such as trituration with a pipette tip to break apart the aggregates. In this study we report on a new chemical dissociation procedure that is efficient, cost effective, reproducible, and much less harmful to murine NSCs than both mechanical and enzymatic techniques. This method, involving the manipulation of environmental pH levels, resulted in 40% higher measured cell densities and 15-20% higher viabilities compared with mechanical dissociation. Moreover, chemical dissociation resulted in the production of significantly less cellular debris. Chemical dissociation was found to have no adverse effects on the long-term proliferation of the NSCs, which retained the ability to proliferate, form neurospheres, self-renew, and exhibit multipotentiality. This chemical method represents a new approach for the dissociation of tissues.