Cell lines, culture media, buffers and detach reagents
HEK293T cells was provided by Obio Technology (Shanghai) Corp., Ltd., and originally purchased from American Type Culture Collection (ATCC, Manassas, VA, CRL-3216). Vero cells was obtained from Cell Bank of Typical Culture Preservation Committee, Chinese Academy of Sciences, and originally obtained from ATCC (CCL-81).
Minimum Essential Medium (MEM, #SH30024.01), Medium 199 (M199, #SH30253.01), DMEM (#SH30243.01), Fetal Bovine Serum (FBS, #SH30084.04IR) were provided by HyClone. HEK293T and Vero cells were grown in above mentioned basal media supplemented with 10% FBS under a 5% CO2 and 37 °C incubator. Dulbecco’s phosphate-buffered saline (D-PBS) without calcium, magnesium (#SH30028.03), 0.25% Trypsin/EDTA (1×, #SH30042.01) and 2.5% Trypsin (10×, #SV30037.01) were provided by HyClone.
Microcarriers
Cytodex-1 microcarrier (#17044803) and Cytodex-1 Gamma microcarrier (#17548702) were ordered from GE Healthcare. The hydration and sterilization process of Cytodex-1 were carried out according to manufacturer’s guide Microcarrier Cell Culture Principles and Methods. Dry Cytodex-1 microcarriers were added into D-PBS (50 ml/g Cytodex-1) at least 4 h for hydration, then washed once using the same volume of D-PBS, and then autoclaved at 121 °C with 30 min. Cytodex-1 microcarriers were used in all HEK293T cell culture processes and Vero cell culture processes in spinner flasks and XDR-50 bioreactor. Cytodex-1 Gamma microcarriers are Gamma-sterilized and ready-to-use for rapid culture start-up. The gamma sterilized microcarriers only require hydration and conditioning, not a heat sterilization process. In this study, Cytodex-1 Gamma microcarriers were applied in Vero cell culture process in XDR-200 bioreactor.
Cell culture medium screening
In this study, media screening experiments for cell growth of HEK293T cells and Vero cells were conducted firstly. Three basal media and one FBS were employed, including MEM, M199, DMEM, and FBS. All the basal media were supplied with 10% FBS and divided into 3 groups, naming as (I) MEM + FBS, (II) M199 + FBS, (III) DMEM + FBS.
HEK293T cells were thawed to the three groups of media individually. After 2 passages adaption, the medium screening process of HEK293T cells was performed in total fifteen T25 flasks. Each T25 flask was inoculated at 2 × 104 cells/cm2 with 5 ml medium, and 5 flasks for each group. After 4-day growth, cell detachment was performed with 1 × Trypsin, and 5 ml corresponding fresh medium without FBS was added into each T25 flasks to suspend the cells. Cell counting was conducted on Beckman Vi-CELL XR Cell Viability Analyzer. The medium screening procedure of Vero cells was same as that of HEK293T cells.
Cell subculture in T-flasks and multilayer flasks
T-Flasks were inoculated with a seeding density of 2 × 104 cells/cm2, and grown in 37 °C with 5% CO2 incubator. When the cultures became confluent after 3–4 days growth, cell detachment procedures were carried out with 1 ml Trypsin/EDTA (1×) per 25 cm2 growth area following a D-PBS wash. Then the flasks or multilayer flasks were placed at 37 °C incubator approximately 3 min. A subculture ratio of 1:4 to 1:6 was most often applied with these cell lines in the T-Flask and multilayer flasks expansion process.
Cell growth in spinner flasks and WAVE 25 bioreactor
Spinner flasks were placed on Micro-Stir Slow Speed Magnetic Stirrers (#W900701-F, Wheaton) in 37 °C with 5% CO2 incubator. Different stir speeds, 60 rpm (Spinner 125, 500 ml), 55 rpm (Spinner 1000 ml), and 50 rpm (Spinner 3000 ml), were set for mixing. Microcarriers Cytodex-1 of 3 g/l were used in all spinners. The spinners were inoculated with a seeding density of 2 ± 0.5 × 105 cells/ml (Rourou et al. 2009). After 2 or 3 days, medium exchanges (30% of total volume) were performed for nutrient replenishment and cell confluence was achieved on day 3 or day 4.
For scale-up process study of Vero cells, WAVE 25 bioreactor as seed train to replace 3 units of spinner 3000 was also investigated (Genzel et al. 2006; Kumar and Starly 2015). The rocking speed setting and rocking motion were very critical for WAVE 25 microcarriers culture. A rocking speed of 12 rpm and a 6° angle was set in the first 2 days post inoculation. The speed was increased to 15 rpm in the following culture days. The rocking motion was set to 100% for a more smoothly rock (Genzel et al. 2006).
Cell morphology and attachment status to microcarriers were evaluated using an inverted microscope (Olympus CKX41, CKX53). Cell density was determined by crystal violet-citric acid method (Kurokawa and Sato 2011) and/or automated cell counter NucleoCounter® NC-200™.
Cell trypsinization on microcarriers and bead-to-bead transfer
When cell confluence was achieved on microcarriers in spinner, the microcarriers were settled by stopping agitation and the supernatant was decanted off carefully in biosafety cabinet (BSC). Microcarriers were then rinsed twice using D-PBS and once with DPBS-1 × EDTA of 30% initial culture volume (iCV) per time. Then 15% iCV of 1 × Trypsin (for HEK293T cells) or 8% iCV of 2 × Trypsin (for Vero cells) were added into the microcarriers. The cell trypsinization process was performed in 37 °C Shaking Water Baths (GLS Aqua 12 Plus, Grant) with gentle mixing every 2 min. After 15–30 min, more than 90% cells were detached from the microcarriers. The microcarriers were settled 2–3 min and the cell supernatants were pumped into a transfer bottle. Washing the microcarriers 2 or 3 times using 20% iCV of DMEM media to elevate cell recovery. Then cell counting was conducted and the cells were prepared to inoculate a new culture. A subcultivation ratio of 1:4 is recommended in microcarrier bead-to-bead expansion processes.
Cell growth comparison on fresh microcarriers and previously populated microcarriers
One of two inoculation methods are typically applied following cell trypsinization as part of the microcarriers scale up process. One method is separating cells from previously populated (spent) beads by mesh or settling microcarriers and only transferring cells to next culture vessel. The advantage of this method is that the cells are evenly distributed across all microcarriers. However, this method will lose some cells, as it is difficult to get 100% of the cells. This will result in decrease in the potential expansion ratio in scale up process. The other method is transferring all the cells and spent beads mixture to next culture vessel following trypsinization. This method has the highest cell utilization and can simplify the operation. However, this may result in uneven cells distribution and differential growth rates on new microcarriers and spent microcarriers.
In this study, two 500 ml spinner flasks were inoculated with Vero cells at a target initial density of 2 × 105 cells/ml. For one spinner, all fresh microcarriers were used and only seed cells (without spent beads) were transferred into the spinner; but for another spinner, cells and partially spent microcarriers (just detached the cells from them) were transferred in. The two spinners were placed in 37 °C with 5% CO2 incubator. Daily samples were taken to observe cell morphology on microcarriers and determine cell density. The microcarriers were settled and 30% of spent media volume was exchanged with fresh media on day 3 and day 4, respectively.
Microcarrier scale-up culture studies in single-use stirred tank bioreactor
Determination of minimum stirring speed for a homogeneous mixing
Microcarriers maybe settle or distribute inhomogeneous because of insufficient mixing at low agitation speed. Uniform suspension and distribution of microcarriers is very important. It not only provides more surface area for cells attachment, but also avoids microenvironments resulting in nutrients and/or dissolved oxygen (DO) depletion. In this study, 10, 15, 20, 25, 30, 35, 40, 45 rpm were set for microcarriers mixing studies in XDR-50 system with culture volume of 20 l to define the minimal agitation speed. Samples were taken out after a minimum of 20 min at each of agitation speeds. 2 ml mixture was added to a Moisture Analyzer (HG63-P, Mettler-Toledo), and the content of microcarriers was detected after 5 min drying to constant weight. Once the mixing speed is sufficient, the microcarriers achieved uniform mixing and this speed is identified as the minimum stirring speed.
Determination of a suitable agitation speed for bioreactor cell culture
A key parameter in bioreactor scale up process is agitation speed. This can be calculated by tip speed or power input per volume. Insufficient agitation speed will result in low mixing efficiency and uneven distribution of cells and nutrients in the bioreactor. However excessively high agitation speed can shear force damage or stress to cells and impact cell growth and production. When choosing an agitation speed for scale up, in addition to the minimum stirring speed, the following two parameters were also considered (Connon 2017; Merten 2015):
Power input per volume
$$ {\text{P}}/{\text{V }} = {\text{ N}}_{\text{p}} {\text{n}}^{ 3} \uprho {\text{ d}}^{ 5} /{\text{V}} $$
(1)
Here P is power input (W/m3), V is volume (m3), Np is impeller’s Newton number or impeller power number, n is agitation speed (r/s), ρ is culture medium density (kg/m3), d is impeller diameter (m).
Kolmogorov eddy size
$$ \upeta = \left[ {\upnu^{ 3} /\left( {{\text{N}}_{\text{p}} {\text{d}}^{ 5} {\text{n}}^{ 3} /{\text{V}}} \right)} \right]^{ 1/ 4} $$
(2)
where η is Kolmogorov eddy size (m), ѵ is kinematic viscosity (m2/s), other symbol’s meanings are same to Eq. (1).
Determination of aeration rate
Another important parameter for cell growth and scale up in stirred tank bioreactors is the aeration rate, which includes the flow rate or sparger rate for compressed air, oxygen, and carbon dioxide. Compressed air and oxygen provide dissolved oxygen for cell growth, and compressed air can also function to regulate culture pCO2 below inhibitory or suboptimal levels. In microcarrier culture processes, high aeration rates can result in significant stress or damage to cells, thereby impacting attachment and growth. This is especially true for the cell types that are not firmly attached to microcarriers, such as HEK293T cells. In order to decrease aeration rate, the mixture aeration (1:4) of compressed air and oxygen were cascaded to DO control in XDR-50 and XDR-200 culture processes. Carbon dioxide is used to adjusted pH value in culture process, however too high CO2 gas flow rate also affect the cell attachment in the microcarrier. In order to reduce the influence of CO2 ventilation on cell attachment in the early stage of cell inoculation, pH value was adjusted to the low limit of pH control, and the CO2 gas flow rate was capped at 0.25 l/min.
Microcarrier scale-up in XDR-50 and XDR-200 bioreactors with HEK293T and Vero cells
HEK293T cells were thawed into T75 flask and subcultivated to T175, then expanded in 125 ml to 3000 ml spinner flasks. The cells from (3) 3000 ml spinner flasks were used to inoculate an XDR-50 bioreactor (Fig. 1). The initial culture volume was 32 l with the inoculum density of 2 × 105 cells/ml and 3 g/l of Cytodex-1 microcarriers. The XDR-50 bioreactor was set to a temperature of 37 °C, and the agitation rate was set to 40 rpm with down pumping mode. Continuous agitation was applied during cell attachment phase and cell growth phase. The pH was maintained at 7.10 ± 0.1 through CO2 sparger and 7.5% NaHCO3 auto pump. DO was maintained at 40 ± 10% of air saturation by cascading air sparger and oxygen sparger. Samples were taken daily from XDR-50 bioreactor to examine cell morphology and concentration, and the metabolites were also analyzed by Nova Profile 400. Glucose concentration was selected as an indicator for media exchange rate. The culture was stopped once cell confluence was achieved on all microcarriers.
Vero cells expansion paradigm was similar to HEK293T cells using the XDR-50 bioreactor. Specifically, 3 g/l Cytodex-1 microcarriers and 32 l culture volume were applied for Vero cell growth in XDR-50 bioreactor, with the initial inoculum of 2 × 105 cells/ml. The agitation rate was set at 40 rpm, which correlates to 6 W/m3 of power input per volume. Other key bioreactor process parameters were set to 37 °C, pH 7.10, DO 40%, and the medium exchange strategies were the same as the HEK293T cell cultivation in XDR-50. The cells were detached by 2 × Trypsin solution in a harvest container once the microcarriers were confluent, then bead-to-bead transfer process was conducted in order to scale to the XDR-200 bioreactor. The Vero cells inoculum and Cytodex-1 microcarrier concentration in XDR-200 bioreactor were similar to the XDR-50, and the culture volume was 106 l. The agitation speed was set to 60 rpm which had a same power input per volume of 6 W/m3, and the eddy size was controlled at 90–95 μm. Oxygen and carbon dioxide sparger gas flow rates were cascaded to DO (40% ± 10%) and pH (7.00 ± 0.20) control, respectively. The maximum sparger flow rate was capped at 1 l/min. The feeding strategy and cell counting was same to XDR-50 and the culture was stopped once the confluence achieved on all microcarriers.
Statistical analysis
Data in this work was analyzed by Microcal Origin 6.0 and presented as mean ± SD. Comparison was performed by Student’s t-test or One-Way ANOVA analysis. P value < 0.05 was considered statistically significant.