The perfusion flow rate can be manipulated depending on the growth and off-line medium analyses to supplement nutrients as necessary with minimal medium fluctuation [29,48]. For example, draining spent medium or wash buffer may also remove some microcarriers with adherent hMSCs and microcarrier removal and cell suspension/concentration steps both involve the loss of some cells. Suspension of microcarriers for cell culture with axial flow impellers. ProCHO5, EXCell Advanced, and PowerCHO2 media all have relatively higher specific productivity when compared to the OptiCHO baseline. Important bioreactor parameters, such as temperature, pH, dissolved oxygen (DO), mixing, feeding strategies and cell counting techniques, are also discussed. This article summarizes key steps involved in the planar culture to microcarrier hMSC manufacturing scheme, from seed train, inoculation, expansion and harvest. In planar culture, cells instinctively adhere the flat surface due to the gravity within a couple of hours after inoculation; contrarily, sufficient cell attachment on microcarriers is determined by cell-bead collisions in addition to adhesion and may take more than one day. The plots indicate relative growth performance with Dynamis having the highest growth rate and ProCHO5 having the lowest. Kaiser S., Jossen V., Schirmaier C., Eibl D., Brill S., van den Bos C., Eibl R. Fluid flow and cell proliferation of mesenchymal adipose-derived stem cells in small-scale, stirred, single-use bioreactors. High throughput screening in microscale bioreactors provides an efficient strategy to identify initial operating parameters. Medium addition simultaneously replenishes nutrients and dilutes metabolic wastes. Within each media, it appears that the exponential phase starts 1 day earlier for the higher inoculation density. Leber J., Barekzai J., Blumenstock M., Pospisil B., Salzig D., Czermak P. Microcarrier choice and bead-to-bead transfer for human mesenchymal stem cells in serum-containing and chemically defined media. Further optimizations to obtain maximum hMSC yields are still needed. Lowell, For example, microcarrier culture requires dynamic flow to suspend microcarriers and homogenize bulk medium. Titer and product quality assays were run on the last day of culture. Tsai A.-C., Liu Y., Ma T. Expansion of human mesenchymal stem cells in fibrous bed bioreactor. This served to confirm that the foam level was being controlled in the rear vessels that could not be inspected visually. Therefore, cell-to-bead ratios from 3 to 6 are commonly used (Table 1). Chemically defined (CD) OptiCHO medium (Thermo Fisher Scientific, Waltham, MA) was the original media in which the cell line was propagated. For clear economic reasons, efficient scaling-up of manufacturing is a desirable endeavor to increase feasibility of allogeneic hMSC therapies [11,12]. Ling WL, In bioreactors, the fouling and coating of foam detection probes is very common, so it is equally important to differentiate between sensor fouling/coating versus foam rising inside the bioreactor. in Bioprocesses The studys conclusion was to retain the mixing as low as required speeds that merely satisfy the mass transfer and avoid microcarrier accumulation in the dead zone [53]. Lin Y.M., Lim J.F.Y., Lee J., Choolani M., Chan J.K.Y., Reuveny S., Oh K.W. Achieving sufficient cell numbers from originating cells requires large in vitro expansion schemes [5]. Downstream technology landscape for large-scale therapeutic cell processing. Our previous studies have delineated the alteration of hMSC properties and therapeutic potencies stemming from micro-environmental differences between planar culture to microcarriers, including the discontinuous surface, convex curvature, microcarrier rigidity, shear stress, collision and aggregation [52,53]. After 6 h of cell adhesion, a continuous stirring was set at 50 rpm. [(accessed on 30 January 2021)]; Antnio M., Fernandes-Platzgummer A., da Silva C.L., Cabral J.M. Kontoravdi C, Cell growth characteristics for media scouting analysis. Dynamis media provided the highest cell growth, having the largest average IVCD of 52 106 cellsd/mL. One is to maintain the temperature of the water jacket of the bioreactor [40,71] or the metal plate attached to the bioreactor bag [44,74] while the other is directly to control the temperature of the bulk culture by measuring the temperature with a thermocouple or a temperature sensor and then to warm the bioreactor with a heating blanket or pad [36,75]. The oxygen mass transfer coefficient is impacted by medium formulations, mixing, energy dissipation, exposure time, etc. Peak MSCAre we there yet? Tsai A.-C., Jeske R., Chen X., Yuan X., Li Y. Antifoam was added to the vessels based on visual observation of foam in culture vessels alone during the media scouting run, although it was noticed that dissolved oxygen measurements, shown in the top of Figure Figure11 for a single vessel, could be used as an indirect way to monitor when foaming occurred. Equilibration buffer used was 25 mM Tris (Diluted from 1 M Tris buffer at pH 7.5(Alfa Aesar, Ward Hill, MA)) with 100 mM NaCl (Thermo Fisher Scientific, Fair Lawn, NJ) at a pH of 7.5. Innertsberger E, The implementation of process analytical technology (PAT) is recommended by the United States Food and Drug Administration (FDA) to clarify processes that aim to facilitate innovation and risk-based regulatory decisions in development, manufacturing and quality assurance [51]. Antifoam chemicals work by reducing the surface tension of the liquid films that form the bubbles within the foam, causing them to more rapidly break down and dissipate. FOIA Qualitative and quantitative demonstration of bead-to-bead transfer with bone marrow-derived human mesenchymal stem cells on microcarriers: Utilising the phenomenon to improve culture performance. Gao F., Chiu S., Motan D., Zhang Z., Chen L., Ji H., Tse H., Fu Q.-L., Lian Q. Mesenchymal stem cells and immuno-modulation: Current status and future prospects. High frequency local variability indicates the occurrence of foam in culture. Gaithersburg, Thermo-responsive microcarriers have also been tested for thermal lifting harvest [87,88]. 10 Mile RoadEast of Inkster, Southfield, MI 48076. The preparation and operation of a bioreactor (also referred to as upstream process in a biotechnological/industrial setting) is comprised of three main steps: expansion (generation of biomass), production (batch, fed-batch, or continuous process), and harvest. HF, shown in the bottom of Figure Figure1.1. Zhao F., Pathi P., Grayson W., Xing Q., Locke B.R., Ma T. Effects of oxygen transport on 3-d human mesenchymal stem cell metabolic activity in perfusion and static cultures: Experiments and mathematical model. The impedance of the material being sensed is used to determine when foam is present. Stirred-tank bioreactors have been widely used in microcarrier suspension culture. In our media scouting analysis we observed that the cells cultured at an inoculation density of 1 106 cells/mL provided marginally higher titers than those cultured at the lower density of 0.5 106 cells/mL, with relative ratios between the same media remaining consistent in comparison to OptiCHO medium. Cells cultured in ProCHO5 showed the lowest overall titer but had a specific productivity comparable to PowerCHO2 and EXCell Advanced with a Q Sen JW, Song K., Yang Y., Wu S., Zhang Y., Feng S., Wang H., Wang Y., Wang L., Liu T. In vitro culture and harvest of BMMSCs on the surface of a novel thermosensitive glass microcarrier. Jing Y, Common operations for hMSC microcarrier-based culture usually implement fed-batch as the feeding strategy to avoid nutrient-related growth inhibition, including medium addition and medium change. Cheng C, Liu L., Yuan W., Wang J. Mechanisms for osteogenic differentiation of human mesenchymal stem cells induced by fluid shear stress. To conclude that the results from the media scouting analysis and the antifoam scouting analysis were reproducible, the experiment was run again with the three media and the three antifoams that showed good specific productivity. OptiCHO was used as the baseline medium for comparison. The method is to count the cell number in a fixed volume and then to convert the number to the sample size and further to the entire culture system. Increased agitation speed can also improve collision frequency; however, reduced contact time during the collision and intensified fluid shear stress due to the higher kinetic energy may ultimately reduce the successful adhesion [63,64]. For example, Harvestainer BioProcess Containers from Thermo Fisher Scientific are designed for separating microcarrier beads from the harvest broth in a closed system at different sizes, including 3 L, 12 L, 25 L and 50 L, which should be able to cover most of the scales developed thus far [86]. In addition to medium replenishment, some studies also tested the addition of fresh, naked microcarriers as an approach to provide existing cells with more surface to expand upon and dilute cell confluency. Yuan Y., Kallos M.S., Hunter C., Sen A. p) of 2 pg/celld. 25 rpm at low volume (1 L) and then increased to 40 rpm at the larger volume (2 L) on Day 3. Cells were harvested and counted using Trypan blue exclusion. Matsuda H, Agarabi CD, Therefore, when the agitation is off, the heating function should be off as well. Improved expansion of human bone marrow-derived mesenchymal stem cells in microcarrier-based suspension culture. The first challenge in developing microcarrier-based hMSC manufacturing is microcarrier selection. 01 h: 200 mL at 50 rpm for 1 min every 15 min. It was assumed that the vessels in back would be foaming around the same time as the replicate vessels in front, so antifoam was added to the vessels at the same time. In this evaluation of antifoams, some more clearly demonstrated cell toxicity, with Antifoam C, EXCell, and SE15 performing most effectively to control foaming while minimizing the total amount of silicone antifoam used. In other words, the observation that cells primarily propagate and proliferate within an individual microcarrier underscores the importance of the initial colonization efficiency that is not a crucial factor in 2D planar culture. WebEffects of anti-foaming agents on biohydrogen production Bioresour Technol. Thus, although these newly evolved bioreactor process parameters improve the scaling-up process at the cell growth stage, they create additional downstream volume-related challenges for microcarrier-based hMSC production. Bethesda, MD 20894, Web Policies HHS Vulnerability Disclosure, Help Furthermore, after a foaming event, material left on the sensor surfaces can cause attenuation of theacoustic signal, preventing switching when the foam level reaches the sensor. Antifoams C, EXCell, and SE15 were capable of providing adequate control of foaming while antifoam 204 and Y30 noticeably stunted cellular growth. Effect of antifoam addition on gasliquid mass transfer in stirred fermenters. Brown MR, As shown in Figure Figure5,5, the specific productivity of cells cultured in ProCHO5, EXCell Advanced, and PowerCHO2 was higher than those of cells cultured in OptiCHO, confirming the media scouting analysis results that these media were an improvement compared to the baseline media. U.S. Food and Drug Administration, Center for Drug Evaluation and Research, Office of Product Quality, Office of Biotechnology Products, Division of Biotechnology Review and Research II, https://creativecommons.org/licenses/by/4.0/, https://bioprocessintl.com/manufacturing/cell-therapies/platform-solutions-cell-therapy-manufacturing/, https://www.fda.gov/regulatory-information/search-fda-guidance-documents/pat-framework-innovative-pharmaceutical-development-manufacturing-and-quality-assurance, https://assets.thermofisher.com/TFS-Assets/BPD/Application-Notes/scalability-harvestainer-app-note.pdf. Sampling ports are used to inject nutrients, water, salts etc. Mizukami A., Chilima T.D.P., Orellana M.D., Neto M.A., Covas D.T., Farid S.S., Swiech K. Technologies for large-scale umbilical cord-derived MSC expansion: Experimental performance and cost of goods analysis. B Appl. This is an open access article under the terms of the, GUID:6807BEC1-5658-4A22-8D20-DF464C507EF6, GUID:1B5E75BA-CF92-40EC-910E-39A01512CC91, GUID:9BB878AF-054D-454D-901B-BFDBC72029FD, GUID:179E7EE3-6C7A-4CFD-B88A-CD8A52427449, GUID:899FB086-E83F-4AD5-9E64-61EADB796A14, GUID:A24BCA13-9930-47ED-8838-F4302DF2A2D4, antifoam, media scouting, microbioreactors, CHO cells, monoclonal antibodies, Evolution and emergence of therapeutic monoclonal antibodies, Mammalian cell protein expression for biopharmaceutical production. Foaming has several undesirable consequences: cell entrapment within the foam, cell damage caused by the bubbles within the foam bursting, reduction of gas transfer rates from the headspace, and over-pressuring of the bioreactor due to clogged vent filters, to name but a few. The interfacial area is dictated by the geometrical parameters, including the shape and dimensions of the bioreactor and sparging bubble size. Loubire C., Sion C., de Isla N., Reppel L., Guedon E., Chevalot I., Olmos E. Impact of the type of microcarrier and agitation modes on the expansion performances of mesenchymal stem cells derived from um-bilical cord. The most critical part of the harvest efficiency is the cell detachment procedure. A related hurdle that complicates multiple steps during the cell harvest processes is incomplete liquid removal. The background was subtracted from the raw signal, as given by Eq. amino acids, vitamins, and trace metals to the media), while other cell lines, configurations, and manufacturing aims may require unique optimization strategies. Res. Routledge SJ, Jung S., Panchalingam K.M., Wuerth R.D., Rosenberg L., Behie L.A. Three media, including ProCHO5, PowerCHO2, and EXCell Advanced, were compared to baseline OptiCHO media in combination with antifoams C, EXCell, and SE15. The .gov means its official. Because of this difficulty in automating foam control, its common to find that the process of antifoam addition is a manual functiona technician sees foam forming and adds chemical to knock it back. The concept is that pumping pure oxygen into the overlay can expedite oxygen dissolvability. This both reduces microcarrier loss during draining and also preserves hMSC-secreted cytokines which are known to enhance expansion. This can give riseto false positives when no foam is present, often from media splashing around in the vessel. There was no considerable difference in specific productivity between the two different inoculation densities; however, the variance was higher at lower inoculation densities for each media. The culture was static for one hour and then start to agitate. Da Silva J.D.S., Severino P., Wodewotzky T.I., Covas D.T., Swiech K., Marti L.C., Suazo C.A.T. Viable cell density and cell viability were measured daily using an automated and integrated ViCell XR cell viability analyzer (Beckman Coulter, Brea, CA). After 6 h of cell adhesion, a continuous stirring was set at 50 rpm. On the contrary, dynamic microcarrier-based cultures containing high cell densities at harvest require more specific controls on pH values. Parts of a stirred tank bioreactor and their function | Cytiva Lund AM, Antifoam C had a higher titer when compared to antifoams EXCell and SE15 in ProCHO 5 and PowerCHO2 but EXCell antifoam performed better in EXCell media in terms of titer (Supplementary Table S2). For example, Shah et al.s experimental results showed that the volumetric oxygen mass transfer coefficient (kLa) increased from 2 to 3.4 h1 when the agitation speed was increased from 6 to 20 rpm in a 2-L gyratory motion bioreactor with an airflow rate of 0.05 vvm, which illustrates that the higher agitation speed improves the oxygen transfer efficiency [80]. Web841 Accesses 62 Citations Metrics Abstract The influence of antifoam agents on the liquid-phase mass transfer coefficient in stirred tank and bubble column bioreactors is studied. The former may have some deviations due to the indirect measurement while the latter requires more attention to understand the relative locations of the temperature sensor and the heating source. Even though colonization efficiency and cell attachment are equally important, few studies have fully evaluated the impacts of both [33,38]. Toward a Clinical-Grade Expansion of Mesenchymal Stem Cells from Human Sources: A Microcarrier-Based Culture system under xeno-free conditions. Nguyen MD, Dept. Problems with foam detection systems are typically related to coating and fouling of sensors, and the resulting generation of false positives. Received 2021 May 2; Accepted 2021 Jul 2. Reinhart D, Antifoaming Agent - an overview | ScienceDirect Topics Tavassoli H., Alhosseini S.N., Tay A., Chan P.P., Oh S.K.W., Warkiani M.E. The formation of foam within bioreactors has been an industry-wide issue for decades. of Chemical Engineering, Samples were washed with PBS and incubated with TrypLE Express at 37 C for 57 min at 650 rpm using Thermomixer confort. UV-VIS-NIR Absorbance and Concentration Measurement. After 1 h, addition of 0.25 L medium was added to reach 3.75 L working volume. Even though there is a diverse array of microcarrier options, they share a similar geometric round shape with a range of 100300 m in diameter. It was observed that ProCHO5, PowerCHO2, and EXCell Advanced medium still performed better than the baseline OptiCHO medium for cell growth and cell specific productivity of antibody, irrespective of the antifoam used. McAfee E., Abraham E. Platform solutions for cell therapy manufacturing. This medium removal step requires no agitation for 10 to 20 min to enable the microcarriers to settle [30,46]. Gentle rocking to distribute the cells, maintaining static culture for overnight prior to addition of medium to working volume. In planar culture, temperature is controlled by the incubator, pH remains balanced by the CO2 concentration with the sodium bicarbonate in the medium, oxygen is supplied by ambient air and nearly complete medium changes provide nutrient replacement and waste removal.
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