Amanda Mizukami

Growth and functional harvesting of human mesenchymal stromal cells cultured on a microcarrier-based system.

Human mesenchymal stromal cells (hMSCs) cells are attractive for applications in tissue engineering and cell therapy. Because of the low availability of hMSCs in tissues and the high doses of hMSCs necessary for infusion, scalable and cost‐effective technologies for in vitro cell expansion are needed to produce MSCs while maintaining their functional, immunophenotypic and cytogenetic characteristics. Microcarrier‐based culture systems are a good alternative to traditional systems for hMSC expansion. The aim of the present study was to develop a scalable bioprocess for the expansion of human bone marrow mesenchymal stromal cells (hBM‐MSCs) on microcarriers to optimize growth and functional harvesting. In general, the results obtained demonstrated the feasibility of expanding hBM‐MSCs using microcarrier technology. The maximum cell concentration (n = 5) was ∼4.82 ± 1.18 × 105 cell mL−1 at day 7, representing a 3.9‐fold increase relative to the amount of inoculated cells. At the end of culture, 87.2% of the cells could be harvested (viability = 95%). Cell metabolism analysis revealed that there was no depletion of important nutrients such as glucose and glutamine during culture, and neither lactate nor ammonia byproducts were formed at inhibitory concentrations. The cells that were recovered after the expansion retained their immunophenotypic and functional characteristics. These results represent an important step toward the implementation of a GMP‐compliant large‐scale production system for hMSCs for cellular therapy.

Combining xanthan and chitosan membranes to multipotent mesenchymal stromal cells as bioactive dressings for dermo-epidermal wounds.

The association between tridimensional scaffolds to cells of interest has provided excellent perspectives for obtaining viable complex tissues in vitro, such as skin, resulting in impressive advances in the field of tissue engineering applied to regenerative therapies. The use of multipotent mesenchymal stromal cells in the treatment of dermo-epidermal wounds is particularly promising due to several relevant properties of these cells, such as high capacity of proliferation in culture, potential of differentiation in multiple skin cell types, important paracrine and immunomodulatory effects, among others. Membranes of chitosan complexed with xanthan may be potentially useful as scaffolds for multipotent mesenchymal stromal cells, given that they present suitable physico-chemical characteristics and have adequate tridimensional structure for the adhesion, growth, and maintenance of cell function. Therefore, the purpose of this work was to assess the applicability of bioactive dressings associating dense and porous chitosan-xanthan membranes to multipotent mesenchymal stromal cells for the treatment of skin wounds. The membranes showed to be non-mutagenic and allowed efficient adhesion and proliferation of the mesenchymal stromal cells in vitro. In vivo assays performed with mesenchymal stromal cells grown on the surface of the dense membranes showed acceleration of wound healing in Wistar rats, thus indicating that the use of this cell-scaffold association for tissue engineering purposes is feasible and attractive.

Stirred tank bioreactor culture combined with serum-/xenogeneic-free culture medium enables an efficient expansion of umbilical cord-derived mesenchymal stem/stromal cells

Mesenchymal stem/stromal cells (MSC) are being widely explored as promising candidates for cell-based therapies. Among the different human MSC origins exploited, umbilical cord represents an attractive and readily available source of MSC that involves a non-invasive collection procedure. In order to achieve relevant cell numbers of human MSC for clinical applications, it is crucial to develop scalable culture systems that allow bioprocess control and monitoring, combined with the use of serum/xenogeneic (xeno)-free culture media. In the present study, we firstly established a spinner flask culture system combining gelatin-based Cultispher(®) S microcarriers and xeno-free culture medium for the expansion of umbilical cord matrix (UCM)-derived MSC. This system enabled the production of 2.4 (±1.1) x10(5) cells/mL (n = 4) after 5 days of culture, corresponding to a 5.3 (±1.6)-fold increase in cell number. The established protocol was then implemented in a stirred-tank bioreactor (800 mL working volume) (n = 3) yielding 115 million cells after 4 days. Upon expansion under stirred conditions, cells retained their differentiation ability and immunomodulatory potential. The development of a scalable microcarrier-based stirred culture system, using xeno-free culture medium that suits the intrinsic features of UCM-derived MSC represents an important step towards a GMP compliant large-scale production platform for these promising cell therapy candidates.

Efficient expansion of mesenchymal stromal cells in a disposable fixed bed culture system

The need for efficient and reliable technologies for clinical‐scale expansion of mesenchymal stromal cells (MSC) has led to the use of disposable bioreactors and culture systems. Here, we evaluate the expansion of cord blood‐derived MSC in a disposable fixed bed culture system. Starting from an initial cell density of 6.0 × 107 cells, after 7 days of culture, it was possible to produce of 4.2(±0.8) × 108 cells, which represents a fold increase of 7.0 (±1.4). After enzymatic retrieval from Fibra‐Cell disks, the cells were able to maintain their potential for differentiation into adipocytes and osteocytes and were positive for many markers common to MSC (CD73, CD90, and CD105). The results obtained in this study demonstrate that MSC can be efficiently expanded in the culture system. This novel approach presents several advantages over the current expansion systems, based on culture flasks or microcarrier‐based spinner flasks and represents a key element for MSC cellular therapy according to GMP compliant clinical‐scale production system.

Characterization of Human AB Serum for Mesenchymal Stromal Cell Expansion

Background: So far, using human blood-derived components appears to be the most efficient and safest approach available for mesenchymal stromal cell (MSC) expansion. In this paper, we report on the characterization of human AB serum (AB HS) produced by using different plasma sources, and its use as an alternative supplement to MSC expansion. Methods: Two plasma sources were used for AB HS production: plasma removed from whole blood after 24 h of collection (PC > 24 h) and plasma, cryoprecipitate reduced (PCryoR). The biochemical profile and quality of the produced AB HS batches were analyzed and their ability to support MSC cell growth after different storage times (0, 3, 6, 9 and 12 months) was evaluated. Results: The two plasma sources used showed similar characteristics regarding biochemical constituents and quality parameters and were effective in promoting MSC growth. MSCs cultured in medium supplemented with 10% AB HS presented similar doubling times and cumulative population doublings when compared to the 10% fetal bovine serum(FBS)-supplemented culture while maintaining immunophenotype, functional features, and cytogenetic profile. Conclusion: Overall, the results indicate that AB HS is an efficient FBS substitute and can be used for at least 12 months after production without impairing cell proliferation and quality.

Expansion strategies for human mesenchymal stromal cells culture under xeno-free conditions

Choosing the culture system and culture medium used to produce cells are key steps toward a safe, scalable, and cost‐effective expansion bioprocess for cell therapy purposes. The use of AB human serum (AB HS) as an alternative xeno‐free supplement for mesenchymal stromal cells (MSC) cultivation has increasingly gained relevance due to safety and efficiency aspects. Here we have evaluated different scalable culture systems to produce a meaningful number of umbilical cord matrix‐derived MSC (UCM MSC) using AB HS for culture medium supplementation during expansion and cryopreservation to enable a xeno‐free bioprocess. UCM MSC were cultured in a scalable planar (compact 10‐layer flasks and roller bottles) and 3‐D microcarrier‐based culture systems (spinner flasks and stirred tank bioreactor). Ten layer flasks and roller bottles enabled the production of 2.6 ± 0.6 × 104 and 1.4 ± 0.3 × 104 cells/cm2. UCM MSC‐based microcarrier expansion in the stirred conditions has enabled the production of higher cell densities (5.5–23.0 × 104 cells/cm2) when compared to planar systems. Nevertheless, due to the moderate harvesting efficiency attained, (80% for spinner flasks and 46.6% for bioreactor) the total cell number recovered was lower than expected. Cells maintained the functional properties after expansion in all the culture systems evaluated. The cryopreservation of cells (using AB HS) was also successfully carried out. Establishing scalable xeno‐free expansion processes represents an important step toward a GMP compliant large‐scale production platform for MSC‐based clinical applications.

A Fully-Closed and Automated Hollow Fiber Bioreactor for Clinical-Grade Manufacturing of Human Mesenchymal Stem/Stromal Cells

The use of mesenchymal stem/stromal cells (MSC) for cellular therapy and regenerative medicine has been boosted by the increasing demand for biological substitutes that can restore, maintain and/or improve the functions of damaged tissues and organs in several diseases. The successful clinical implementation of MSC-based therapies must address the need for ex-vivo expansion due to the low frequency of these cells in the available tissues and the high doses (1-100 × 106 cells/kg patient) required for an infusion. The manufacturing of high cell doses/lot will be possible through the use of bioreactors that present several advantages when compared to the traditional flask-based methods: traceability, scalability, reduced manual handling and easy monitorability and control of culture parameters. Moreover, the use of defined serum-/xenogeneic(xeno)-free culture medium formulations could result in substantial improvements for MSC production in terms of cell reproducibility, stability and quality, as well as safety, while ensuring the approval by regulatory agencies. The combination of bioreactors and xeno-free culture conditions enable the large-scale production of clinical-grade MSC, contributing towards the progression of this medicinal therapeutic product into the clinics [1]. Hollow fiber bioreactors are considered a viable option for MSC expansion, due to their relatively homogeneous culture environment, excellent mass transfer properties and low level of shear stress. Here we demonstrate, for the first time, the use of the Quantum® Cell Expansion System (Terumo BCT) for the effective ex-vivo expansion of adipose tissue-derived MSC (AT MSC) under xeno-free conditions. Adipose tissue samples from liposuction surgery were provided after written informed consent by Hospital das Clínicas, University of São Paulo, Ribeirão Preto, through the ethic project 375,415/2013. In our experiments, AT MSC (2 independent donors), previously isolated using xeno-free culture medium, were expanded (seeding density: 3000 cells/cm2) on tissue flasks using Dulbecco ́s modified Eagle ́s medium (DMEM) supplemented with 5% (v/v) xeno-free fibrinogen-depleted human platelet lysate (FDhPL) (UltraGROTM-PURE, AventaCell BioMedical Corp.) and 1% (v/v) antibiotic–antimycotic. Then, 30 × 106 AT MSC (passage 4) were inoculated (1000 cells/ cm2) into the intracapillary space (ICS) (previously coated with fibronectin to promote adhesion) of the Quantum® bioreactor and allowed to attach for 24 h. Afterwards, cells were fed through a continuous flow of culture medium in the extracapillary space (ECS) (initial rate of 0.1 mL/min) and perfusion rate was increased (0.1–0.6 mL/min) when lactate concentration reached values potentially inhibitory (8–9 mM). After 5 days of cultivation, cells were harvested by two enzymatic dissociation cycles (TrypLETM Select, 10x), being the final cell number and viability determined using the Trypan Blue exclusion method. Glucose, lactate and glutamine were determined in the culture supernatant * Kamilla Swiech kamilla@fcfrp.usp.br

Platforms for Recombinant Therapeutic Glycoprotein Production

The majority of FDA-approved biology-derived products are recombinant glycoproteins. These proteins have been used for the treatment of several diseases, with numerous products currently approved for clinical use. The choice of the expression system is a key step toward a successful functional protein production, since glycosylation influences yield, pharmacokinetics, biological activity, and immunogenicity. This chapter covers the general aspects of therapeutic recombinant glycoproteins and the platforms that are being employed for their production.

Mesenchymal Stromal Cells: From Discovery to Manufacturing and Commercialization

Over the last decades, mesenchymal stromal cells (MSC) have been the focus of intense research by academia and industry due to their unique features. MSC can be easily isolated and expanded through in vitro culture by taking full advantage of their self-renewing capacity. In addition, MSC exert immunomodulatory effects and can be differentiated into various lineages, which makes them highly attractive for clinical applications in cell-based therapies. In this review, we attempt to provide a brief historical overview of MSC discovery, characterization, and the first clinical studies conducted. The current MSC manufacturing platforms are reviewed with special attention regarding the use of bioreactors for the production of GMP-compliant clinically relevant cell numbers. The first commercial MSC-based products are also addressed, as well as the remaining challenges to the widespread use of MSC-derived products.