Leo A. Behie

Ex Vivo Expansion of Human Mesenchymal Stem Cells in Defined Serum-Free Media

Human mesenchymal stem cells (hMSCs) are presently being evaluated for their therapeutic potential in clinical studies to treat various diseases, disorders, and injuries. To date, early-phase studies have indicated that the use of both autologous and allogeneic hMSCs appear to be safe; however, efficacy has not been demonstrated in recent late-stage clinical trials. Optimized cell bioprocessing protocols may enhance the efficacy as well as safety of hMSC therapeutics. Classical media used for generating hMSCs are typically supplemented with ill-defined supplements such as fetal bovine serum (FBS) or human-sourced alternatives. Ideally, culture media are desired to have well-defined serum-free formulations that support the efficient production of hMSCs while maintaining their therapeutic and differentiation capacity. Towards this objective, we review here current cell culture media for hMSCs and discuss medium development strategies.

Axial dispersion in the three-dimensional mixing of particles in a rotating drum reactor

Horizontal drum reactors are widely used in industry for the processing of granular material. They are ideally suited for chemical processes that require high temperatures at near-atmospheric pressure. However, the complexities of these reactors have resulted in empirical design procedures that lead to very conservative and costly reactors. This study first reviews critically the extensive literature on experimental results obtained on rotary kilns (without flights) and proposes new design equations for the axial-dispersion coefficient in terms of rotational speed, degree of fill, drum diameter, and particle diameter. A total of 179 data points from the literature, encompassing both the batch and the continuous operational modes, yielded design correlations for slumping, rolling/cascading and cataracting bed behaviours. Additionally, new measurements were made on a pilot-scale rotary drum by tracking a single radioactive particle (emitting gamma-rays) using a battery of nine scintillation counters; these data confirmed the correctness of the proposed design correlations.

Inoculation and growth conditions for high-cell-density expansion of mammalian neural stem cells in suspension bioreactors

Inoculation and growth conditions for the large-scale expansion of mammalian neural stem cells (NSC) have been determined. We examined suspension culture bioreactors of murine NSC, and concluded that the oxygen level should be kept high (20%), and the osmolarity of the medium should be kept low (below 400 mOsm/kg). The pH of the medium was found to have a large effect on cell proliferation, and the best growth characteristics were obtained within an optimum pH range of 7. 1 to 7.5. The inoculation conditions were also seen to have a large effect not only on the growth characteristics, but also on the number of cells that die in the initial stages of the culture. For large expansion of cells, low inoculum levels (10(4) cells/mL) and single-cell suspensions proved superior, whereas, for fast expansion of cells, higher inoculum levels (10(5) cells/mL) and spheroid inoculum forms were preferred. The inoculum temperature of the medium did not have a large effect on growth characteristics, but the pH greatly influenced cell proliferation. Inoculum pH levels should also be kept between 7.1 and 7.5. If these protocols are followed, high multiplication ratios and viabilities can be obtained in a 5-day batch suspension culture bioreactor run. A large number of cells could then be used in animal models for testing of neural drugs and in research and development toward cures for neurodegenerative disorders such as multiple sclerosis (MS) and Huntingtons and Parkinsons disease. The results presented here also point the way toward studies on in vitro expansion of human neural stem cells.

High-level expression of secreted glycoproteins in transformed lepidopteran insect cells using a novel expression vector

An expression cassette for continuous high-level expression of secreted glycoproteins by transformed lepidopteran insect cells has been developed as an alternative to baculovirus and mammalian cell expression systems. The expression cassette utilizes the promoter of the silkmoth cytoplasmic actin gene to drive expression from foreign gene sequences, and also contains the ie-1 transactivator gene and the HR3 enhancer region of BmNPV to stimulate gene expression. Using an antibiotic-resistance selection scheme, we have cloned a Bm5 (silkmoth) cell line overexpressing the secreted glycoprotein juvenile hormone esterase (JHE-KK) at levels of 190 mg/L in batch suspension cultures. A baculovirus (AcNPV) expressing the same gene under the control of the p10 promoter of AcNPV produced only 4 mg/L active JHE in static cultures of infected Sf21 cells. A cloned Bm5 cell line overexpressing a soluble isoform of the alpha-subunit of the granulocyte-macrophage colony stimulating factor receptor (solGMRalpha) was also generated and produced five times more solGMRalpha in static cultures than a cloned BHK cell line obtained by transformation with a recombinant expression cassette utilizing the human cytomegalovirus (CMV) enhancer-promoter system. Finally, we show that recombinant protein expression levels in transformed Bm5 cells remain high in serum-free media, that expression is stable even in the absence of antibiotic selection, and that lepidopteran cells other than Bm5 may be used equally efficiently with this new expression cassette for producing recombinant proteins.

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.

Ultrapyrolysis of n-hexadecane in a novel micro-reactor

A novel reaction system, which provides better control compared with the tubular flow reactors of previous researchers, was employed to study the ultrapyrolysis kinetics of n-hexadecane, a gas oil model compound. Ultrapyrolysis, or ultra rapid pyrolysis, refers to thermal cracking under conditions of high temperature, very short reaction time, high heating rate and rapid product quench. Modern-day ‘millisecond’ furnaces, operating under such conditions, have demonstrated significantly higher ethylene yields due to improved selectivity. The automated micro-reaction system employed a Curie point pyrolyser to rapidly heat microgram n-hexadecane samples to high temperature. The Curie point phenomenon ensured that a reproducible and well-defined temperature was attained. A rapid direct quench system was used to stop the reactions and transfer the products to the analyser. n-Hexadecane was pyrolysed at 576–842 °C for 100–3200 ms. Peak ethylene production (28 wt%) was exhibited at ultrapyrolytic conditions of 842 °C and 500 ms. A first-order kinetic analysis performed on the pyrolysis data yielded an activation energy of 39.4 kcal mol−1.

SOLIDS CIRCULATION IN SPOUTED AND SPOUT-FLUID BEDS WITH DRAFT-TUBES

A 20 cm semi-cylindrical spout-fluid bed with a draft-tube has been used to investigate the effect of various physical parameters on the solids circulation rate, A new method has been developed by which these data can be easily and accurately collected. Equations are presented which can be used to predict the maximum attainable solids circulation rate and the spouting velocity required to achieve this maximum for the unit operating in spouting mode. The circulation trends are shown to be a direct result of the phenomena of entrainment and jetting occurring in the entrainment zone investigated visually with a video camera. Preliminary gas bypassing experiments were also performed which show the ability to almost eliminate spout gas bypassing to the annulus with appropriate auxiliary gas addition.

Large-scale production of human mesenchymal stem cells for clinical applications

Human mesenchymal stem cells (hMSCs) have many potential applications in tissue engineering and regenerative medicine. Currently, hMSCs are generated through conventional static adherent cultures in the presence of fetal bovine serum (FBS) for clinical applications (e.g., multiple sclerosis). However, these methods are not appropriate to meet the expected future demand for quality‐assured hMSCs for human therapeutic use. Hence, it is imperative to develop an effective hMSC production system, which should be controllable, reproducible, and scalable. To this end, efforts have been made by several international research groups to develop (i) alternative media either by replacing FBS with human‐sourced supplements (such as human serum or platelet lysate) or by identifying defined serum‐free formulations consisting of key growth/attachment factors, and (ii) controlled bioreactor protocols. In this regard, we review here current hMSC production technologies and future perspectives toward efficient methods for the generation of clinically relevant numbers of hMSC therapeutics.

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.