Mesenchymal and induced pluripotent stem cell-based therapeutics: a comparison.

Stem cell-based cell therapeutics and especially those based on human mesenchymal stem cells (hMSCs) and induced pluripotent stem cells (hiPSCs) are said to have enormous developmental potential in the coming years. Their applications range from the treatment of orthopedic disorders and cardiovascular diseases to autoimmune diseases and even cancer. However, while more than 27 hMSC-derived therapeutics are currently commercially available, hiPSC-based therapeutics have yet to complete the regulatory approval process. Based on a review of the current commercially available hMSC-derived therapeutic products and upcoming hiPSC-derived products in phase 2 and 3, this paper compares the cell therapy manufacturing process between these two cell types. Moreover, the similarities as well as differences are highlighted and the resulting impact on the production process discussed. Here, emphasis is placed on (i) hMSC and hiPSC characteristics, safety, and ethical aspects, (ii) their morphology and process requirements, as well as (iii) their 2- and 3-dimensional cultivations in dependence of the applied culture medium and process mode. In doing so, also downstream processing aspects are covered and the role of single-use technology is discussed. KEY POINTS: • Mesenchymal and induced pluripotent stem cells exhibit distinct behaviors during cultivation • Single-use stirred bioreactor systems are preferred for the cultivation of both cell types • Future research should adapt and modify downstream processes to available single-use devices.

An overview of drive systems and sealing types in stirred bioreactors used in biotechnological processes.

No matter the scale, stirred tank bioreactors are the most commonly used systems in biotechnological production processes. Single-use and reusable systems are supplied by several manufacturers. The type, size, and number of impellers used in these systems have a significant influence on the characteristics and designs of bioreactors. Depending on the desired application, classic shaft-driven systems, bearing-mounted drives, or stirring elements that levitate freely in the vessel may be employed. In systems with drive shafts, process hygiene requirements also affect the type of seal used. For sensitive processes with high hygienic requirements, magnetic-driven stirring systems, which have been the focus of much research in recent years, are recommended. This review provides the reader with an overview of the most common agitation and seal types implemented in stirred bioreactor systems, highlights their advantages and disadvantages, and explains their possible fields of application. Special attention is paid to the development of magnetically driven agitators, which are widely used in reusable systems and are also becoming more and more important in their single-use counterparts.Key Points• Basic design of the most frequently used bioreactor type: the stirred tank bioreactor• Differences in most common seal types in stirred systems and fields of application• Comprehensive overview of commercially available bioreactor seal types• Increased use of magnetically driven agitation systems in single-use bioreactors.

Universities of Applied Sciences.

When the SARS-CoV-2 pandemic started,[1] science came to the immediate attention of the broad public. People and politicians were hanging on every word of medical doctors, virologists, molecular biologists, data scientists and many others in the hope of finding other protective measures than those used for centuries such as basic hygiene, distance, or quarantine. Here, at the Institute of Chemistry and Biotechnology at the Zurich University of Applied Sciences (ZHAW) we were also willing to provide scientific solutions to overcome the pandemic. Together with our partners from industry, we contributed to the development of a Swiss vaccine, are working on filters for active ventilated full protective suits and are developing tests to show the efficacy and safety of an active antiviral textile that allows controlled virus inactivation through an electrochemical reaction by applying a small current.

Manufacturing human mesenchymal stem cells at clinical scale: process and regulatory challenges.

Human mesenchymal stem cell (hMSC)-based therapies are of increasing interest in the field of regenerative medicine. As economic considerations have shown, allogeneic therapy seems to be the most cost-effective method. Standardized procedures based on instrumented single-use bioreactors have been shown to provide billion of cells with consistent product quality and to be superior to traditional expansions in planar cultivation systems. Furthermore, under consideration of the complex nature and requirements of allogeneic hMSC-therapeutics, a new equipment for downstream processing (DSP) was successfully evaluated. This mini-review summarizes both the current state of the hMSC production process and the challenges which have to be taken into account when efficiently producing hMSCs for the clinical scale. Special emphasis is placed on the upstream processing (USP) and DSP operations which cover expansion, harvesting, detachment, separation, washing and concentration steps, and the regulatory demands.

Plant cell culture technology in the cosmetics and food industries: current state and future trends.

The production of drugs, cosmetics, and food which are derived from plant cell and tissue cultures has a long tradition. The emerging trend of manufacturing cosmetics and food products in a natural and sustainable manner has brought a new wave in plant cell culture technology over the past 10 years. More than 50 products based on extracts from plant cell cultures have made their way into the cosmetics industry during this time, whereby the majority is produced with plant cell suspension cultures. In addition, the first plant cell culture-based food supplement ingredients, such as Echigena Plus and Teoside 10, are now produced at production scale. In this mini review, we discuss the reasons for and the characteristics as well as the challenges of plant cell culture-based productions for the cosmetics and food industries. It focuses on the current state of the art in this field. In addition, two examples of the latest developments in plant cell culture-based food production are presented, that is, superfood which boosts health and food that can be produced in the lab or at home.

Novel probes for pH and dissolved oxygen measurements in cultivations from millilitre to benchtop scale.

pH value and the concentration of dissolved oxygen (DO) are key parameters to monitor and control cell growth in cultivation studies. Reliable, robust and accurate methods to measure these parameters in cultivation systems in real time guarantee high product yield and quality. This mini-review summarises the current state of the art of pH and DO sensors that are applied to bioprocesses from millilitre to benchtop scale by means of a short introduction on measuring principles and selected applications. Special emphasis is placed on single-use bioreactors, which have been increasingly employed in bioprocess development and production in recent years. Working principles, applications and the particular requirements of sensors in these cultivation systems are given. In such processes, optical sensors for pH and DO are often preferred to electrochemical probes, as they allow semi-invasive measurements and can be miniaturised to micrometre scale or lower. In addition, selected measuring principles of novel sensing technologies for pH and DO are discussed. These include solid-state sensors and miniaturised devices that are not yet commercially available, but show promising characteristics for possible use in bioprocesses in the near future.

Disposable Bioreactors Used in Process Development and Production Processes with Plant Cell and Tissue Cultures.

The bioreactor is the centerpiece of the upstream processing in any biotechnological production process. Its design, the cultivation parameters, the production cell line, and the culture medium all have a major influence on the efficiency of the process and the result of the cultivation. Disposable bioreactors have been used for the past 20 years, playing a major role in process development and commercial production of high-value substances at medium scales.Our review deals with scalable, disposable bioreactors that have proven to be useful for the cultivation of plant cell and tissue cultures. Based on the definitions of terms and a categorization approach, the most commonly used, commercially available, disposable bioreactor types are presented below. The focus is on wave-mixed, stirred, and orbitally shaken bioreactors. In addition to their instrumentation and bioengineering characteristics, cultivation results are discussed, and emerging trends for the development of disposable bioreactors for plant cell and tissue cultures are also addressed.

Improvement of HEK293 Cell Growth by Adapting Hydrodynamic Stress and Predicting Cell Aggregate Size Distribution.

HEK293 is a widely used cell line in the fields of research and industry. It is assumed that these cells are sensitive to hydrodynamic stress. The aim of this research was to use particle image velocimetry validated computational fluid dynamics (CFD) to determine the hydrodynamic stress in both shake flasks, with and without baffles, and in stirred Minifors 2 bioreactors to evaluate its effect on the growth and aggregate size distribution of HEK293 suspension cells. The HEK FreeStyleTM 293-F cell line was cultivated in batch mode at different specific power inputs (from 63 W m-3 to 451 W m-3), whereby ≈60 W m-3 corresponds to the upper limit, which is what has been typically described in published experiments. In addition to the specific growth rate and maximum viable cell density VCDmax, the cell size distribution over time and cluster size distribution were investigated. The VCDmax of (5.77±0.02)·106cellsmL-1 was reached at a specific power input of 233 W m-3 and was 23.8% higher than the value obtained at 63 W m-3 and 7.2% higher than the value obtained at 451 W m-3. No significant change in the cell size distribution could be measured in the investigated range. It was shown that the cell cluster size distribution follows a strict geometric distribution whose free parameter p is linearly dependent on the mean Kolmogorov length scale. Based on the performed experiments, it has been shown that by using CFD-characterised bioreactors, the VCDmax can be increased and the cell aggregate rate can be precisely controlled.

Chemically Defined, Xeno-Free Expansion of Human Mesenchymal Stem Cells (hMSCs) on Benchtop-Scale Using a Stirred Single-Use Bioreactor.

In recent years, the use of hMSCs, which may be isolated from adipose tissue among others, for the treatment of diseases has increased significantly. The cell quantities required for such therapeutic approaches, between 1012 and 1013, have thus far been predominantly produced using commercially available multi-tray systems, such as the Cell Factory (Thermo Fisher Scientific) or HYPERStack (Corning), which can be purchased with up to 40 layers. However, the handling of these planar multilayer systems is difficult, and process monitoring opportunities remain limited. Here, automated stirred single-use bioreactors provide a viable alternative to the time-consuming multiplication of cells using such planar systems, while still managing to achieve the desired clinically relevant quantities. In these stirred single-use systems, adherent cells are predominantly cultivated in suspension up to pilot scale using carrier materials, also referred to as microcarriers (MCs).This chapter describes the steps which need to be realized to guarantee successful hMSC expansion within a stirred single-use bioreactor (Eppendorf's BioBLU® 0.3c) operated using MCs under serum- and xeno-free conditions at benchtop scale. The cultivations were performed using an immortalized human adipose-derived mesenchymal stem cell (hASC) line, hence referred to as hASC52telo, and a new chemically defined, xeno-free medium, hence referred to as the UrSuppe formulation. Spinner flask cultivations were performed under comparable process conditions.

Improved Time Resolved KPI and Strain Characterization of Multiple Hosts in Shake Flasks Using Advanced Online Analytics and Data Science.

Shake flasks remain one of the most widely used cultivation systems in biotechnology, especially for process development (cell line and parameter screening). This can be justified by their ease of use as well as their low investment and running costs. A disadvantage, however, is that cultivations in shake flasks are black box processes with reduced possibilities for recording online data, resulting in a lack of control and time-consuming, manual data analysis. Although different measurement methods have been developed for shake flasks, they lack comparability, especially when changing production organisms. In this study, the use of online backscattered light, dissolved oxygen, and pH data for characterization of animal, plant, and microbial cell culture processes in shake flasks are evaluated and compared. The application of these different online measurement techniques allows key performance indicators (KPIs) to be determined based on online data. This paper evaluates a novel data science workflow to automatically determine KPIs using online data from early development stages without human bias. This enables standardized and cost-effective process-oriented cell line characterization of shake flask cultivations to be performed in accordance with the process analytical technology (PAT) initiative. The comparison showed very good agreement between KPIs determined using offline data, manual techniques, and automatic calculations based on multiple signals of varying strengths with respect to the selected measurement signal.

Food ingredients and food made with plant cell and tissue cultures: State-of-the art and future trends.

Climate change and an increasing world population means traditional farming methods may not be able to meet the anticipated growth in food demands. Therefore, alternative agricultural strategies should be considered. Here, plant cell and tissue cultures (PCTCs) may present a possible solution, as they allow for controlled, closed and sustainable manufacturing of extracts which have been or are still being used as colorants or health food ingredients today. In this review we would like to highlight developments and the latest trends concerning commercial PCTC extracts and their use as food ingredients or even as food. The commercialization of PCTC-derived products, however, requires not only regulatory approval, but also outstanding product properties or/and a high product titer. If these challenges can be met, PCTCs will become increasingly important for the food sector in coming years.

Numerical Methods for the Design and Description of In Vitro Expansion Processes of Human Mesenchymal Stem Cells.

Human mesenchymal stem cells (hMSCs) are a valuable source of cells for clinical applications (e.g., treatment of acute myocardial infarction or inflammatory diseases), especially in the field of regenerative medicine. However, for autologous (patient-specific) and allogeneic (off-the-shelf) hMSC-based therapies, in vitro expansion is necessary prior to the clinical application in order to achieve the required cell numbers. Safe, reproducible, and economic in vitro expansion of hMSCs for autologous and allogeneic therapies can be problematic because the cell material is restricted and the cells are sensitive to environmental changes. It is beneficial to collect detailed information on the hydrodynamic conditions and cell growth behavior in a bioreactor system, in order to develop a so called "Digital Twin" of the cultivation system and expansion process. Numerical methods, such as Computational Fluid Dynamics (CFD) which has become widely used in the biotech industry for studying local characteristics within bioreactors or kinetic growth modelling, provide possible solutions for such tasks.In this review, we will present the current state-of-the-art for the in vitro expansion of hMSCs. Different numerical tools, including numerical fluid flow simulations and cell growth modelling approaches for hMSCs, will be presented. In addition, a case study demonstrating the applicability of CFD and kinetic growth modelling for the development of an microcarrier-based hMSC process will be shown.

Cellular Agriculture: Opportunities and Challenges.

Cellular agriculture is the controlled and sustainable manufacture of agricultural products with cells and tissues without plant or animal involvement. Today, microorganisms cultivated in bioreactors already produce egg and milk proteins, sweeteners, and flavors for human nutrition as well as leather and fibers for shoes, bags, and textiles. Furthermore, plant cell and tissue cultures provide ingredients that stimulate the immune system and improve skin texture, with another precommercial cellular agriculture product, in vitro meat, currently receiving a great deal of attention. All these approaches could assist traditional agriculture in continuing to provide for the dietary requirements of a growing world population while freeing up important resources such as arable land. Despite early successes, challenges remain and are discussed in this review, with a focus on production processes involving plant and animal cell and tissue cultures.

Disposable bioreactors: the current state-of-the-art and recommended applications in biotechnology.

Disposable bioreactors have increasingly been incorporated into preclinical, clinical, and production-scale biotechnological facilities over the last few years. Driven by market needs, and, in particular, by the developers and manufacturers of drugs, vaccines, and further biologicals, there has been a trend toward the use of disposable seed bioreactors as well as production bioreactors. Numerous studies documenting their advantages in use have contributed to further new developments and have resulted in the availability of a multitude of disposable bioreactor types which differ in power input, design, instrumentation, and scale of the cultivation container. In this review, the term "disposable bioreactor" is defined, the benefits and constraints of disposable bioreactors are discussed, and critical phases and milestones in the development of disposable bioreactors are summarized. An overview of the disposable bioreactors that are currently commercially available is provided, and the domination of wave-mixed, orbitally shaken, and, in particular, stirred disposable bioreactors in animal cell-derived productions at cubic meter scale is reported. The growth of this type of reactor system is attributed to the recent availability of stirred disposable benchtop systems such as the Mobius CellReady 3 L Bioreactor. Analysis of the data from computational fluid dynamic simulation studies and first cultivation runs confirms that this novel bioreactor system is a viable alternative to traditional cell culture bioreactors at benchtop scale.

Innovative, non-stirred bioreactors in scales from milliliters up to 1000 liters for suspension cultures of cells using disposable bags and containers--a Swiss contribution.

Innovative mixing principles in bioreactors, for example using the rocking of a platform to induce a backwards and forwards 'wave', or using orbital shaking to generate a 'wave' that runs round in a cylindrical container, have proved to be successful for the suspension cultures of cells, especially when combined with disposable materials. This article presents an overview of the engineering characteristics when these new principles are applied in bioreactors, and case studies covering scales of operation from milliliters to 1000 liters.

How to Produce mAbs in a Cube-Shaped Stirred Single-Use Bioreactor at 200 L Scale.

Single-use bioreactors have increasingly been used in recent years, for both research and development as well as industrial production, especially in mammalian cell-based processes. Among the numerous single-use bioreactors available today, wave-mixed bags and stirred systems dominate. Wave-mixed single-use bioreactors are the system of choice for inoculum production, while stirred single-use bioreactors are most often preferred for antibody expression. For this reason, the present chapter describes protocols instructing the reader to use the wave-mixed BIOSTAT® RM 50 for cell expansion and to produce a monoclonal antibody (mAb) in Pall's Allegro™ STR 200 at pilot scale for the first time. All methods described are based on a Chinese hamster ovary (CHO) suspension cell line expressing a recombinant immunoglobulin G (IgG).

Growth Behavior of Human Adipose Tissue-Derived Stromal/Stem Cells at Small Scale: Numerical and Experimental Investigations.

Human adipose tissue-derived stromal/stem cells (hASCs) are a valuable source of cells for clinical applications, especially in the field of regenerative medicine. Therefore, it comes as no surprise that the interest in hASCs has greatly increased over the last decade. However, in order to use hASCs in clinically relevant numbers, in vitro expansion is required. Single-use stirred bioreactors in combination with microcarriers (MCs) have shown themselves to be suitable systems for this task. However, hASCs tend to be less robust, and thus, more shear sensitive than conventional production cell lines for therapeutic antibodies and vaccines (e.g., Chinese Hamster Ovary cells CHO, Baby Hamster Kidney cells BHK), for which these bioreactors were originally designed. Hence, the goal of this study was to investigate the influence of different shear stress levels on the growth of humane telomerase reversed transcriptase immortalized hASCs (hTERT-ASC) and aggregate formation in stirred single-use systems at the mL scale: the 125 mL (= SP100) and the 500 mL (= SP300) disposable Corning® spinner flask. Computational fluid dynamics (CFD) simulations based on an Euler⁻Euler and Euler⁻Lagrange approach were performed to predict the hydrodynamic stresses (0.06⁻0.87 Pa), the residence times (0.4⁻7.3 s), and the circulation times (1.6⁻16.6 s) of the MCs in different shear zones for different impeller speeds and the suspension criteria (Ns1u, Ns1). The numerical findings were linked to experimental data from cultivations studies to develop, for the first time, an unstructured, segregated mathematical growth model for hTERT-ASCs. While the 125 mL spinner flask with 100 mL working volume (SP100) provided up to 1.68.105 hTERT-ASC/cm² (= 0.63 × 106 living hTERT-ASCs/mL, EF 56) within eight days, the peak living cell density of the 500 mL spinner flask with 300 mL working volume (SP300) was 2.46 × 105 hTERT-ASC/cm² (= 0.88 × 106 hTERT-ASCs/mL, EF 81) and was achieved on day eight. Optimal cultivation conditions were found for Ns1u < N < Ns1, which corresponded to specific power inputs of 0.3⁻1.1 W/m³. The established growth model delivered reliable predictions for cell growth on the MCs with an accuracy of 76⁻96% for both investigated spinner flask types.

Power Input Measurements in Stirred Bioreactors at Laboratory Scale.

The power input in stirred bioreactors is an important scaling-up parameter and can be measured through the torque that acts on the impeller shaft during rotation. However, the experimental determination of the power input in small-scale vessels is still challenging due to relatively high friction losses inside typically used bushings, bearings and/or shaft seals and the accuracy of commercially available torque meters. Thus, only limited data for small-scale bioreactors, in particular single-use systems, is available in the literature, making comparisons among different single-use systems and their conventional counterparts difficult. This manuscript provides a protocol on how to measure power inputs in benchtop scale bioreactors over a wide range of turbulence conditions, which can be described by the dimensionless Reynolds number (Re). The aforementioned friction losses are effectively reduced by the use of an air bearing. The procedure on how to set up, conduct and evaluate a torque-based power input measurement, with special focus on cell culture typical agitation conditions with low to moderate turbulence (100 < Re < 2·104), is described in detail. The power input of several multi-use and single-use bioreactors is provided by the dimensionless power number (also called Newton number, P0), which is determined to be in the range of P0 ≈ 0.3 and P0 ≈ 4.5 for the maximum Reynolds numbers in the different bioreactors.

Development of a method for reliable power input measurements in conventional and single-use stirred bioreactors at laboratory scale.

Power input is an important engineering and scale-up/down criterion in stirred bioreactors. However, reliably measuring power input in laboratory-scale systems is still challenging. Even though torque measurements have proven to be suitable in pilot scale systems, sensor accuracy, resolution, and errors from relatively high levels of friction inside bearings can become limiting factors at smaller scales. An experimental setup for power input measurements was developed in this study by focusing on stainless steel and single-use bioreactors in the single-digit volume range. The friction losses inside the air bearings were effectively reduced to less than 0.5% of the measurement range of the torque meter. A comparison of dimensionless power numbers determined for a reference Rushton turbine stirrer (NP = 4.17 ± 0.14 for fully turbulent conditions) revealed good agreement with literature data. Hence, the power numbers of several reusable and single-use bioreactors could be determined over a wide range of Reynolds numbers between 100 and >104. Power numbers of between 0.3 and 4.5 (for Re = 104) were determined for the different systems. The rigid plastic vessels showed similar power characteristics to their reusable counterparts. Thus, it was demonstrated that the torque-based technique can be used to reliably measure power input in stirred reusable and single-use bioreactors at the laboratory scale.

Theoretical and Practical Issues That Are Relevant When Scaling Up hMSC Microcarrier Production Processes.

The potential of human mesenchymal stem cells (hMSCs) for allogeneic cell therapies has created a large amount of interest. However, this presupposes the availability of efficient scale-up procedures. Promising results have been reported for stirred bioreactors that operate with microcarriers. Recent publications focusing on microcarrier-based stirred bioreactors have demonstrated the successful use of Computational Fluid Dynamics (CFD) and suspension criteria (N S1u , N S1) for rapidly scaling up hMSC expansions from mL- to pilot scale. Nevertheless, one obstacle may be the formation of large microcarrier-cell-aggregates, which may result in mass transfer limitations and inhomogeneous distributions of stem cells in the culture broth. The dependence of microcarrier-cell-aggregate formation on impeller speed and shear stress levels was investigated for human adipose derived stromal/stem cells (hASCs) at the spinner scale by recording the Sauter mean diameter (d 32) versus time. Cultivation at the suspension criteria provided d 32 values between 0.2 and 0.7 mm, the highest cell densities (1.25 × 10(6) cells mL(-1) hASCs), and the highest expansion factors (117.0 ± 4.7 on day 7), while maintaining the expression of specific surface markers. Furthermore, suitability of the suspension criterion N S1u was investigated for scaling up microcarrier-based processes in wave-mixed bioreactors for the first time.