Development of an Animal-Derived Component-Free Medium for Spodoptera frugiperda (Sf9) Cells Using Response Surface Methodology

doi:10.21203/rs.3.rs-2437075/v1

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Development of an Animal-Derived Component-Free Medium for Spodoptera frugiperda (Sf9) Cells Using Response Surface Methodology

https://doi.org/10.21203/rs.3.rs-2437075/v1

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Spodoptera frugiperda (Sf9) insect cells have been widely used for recombinant protein production. Currently, serum-free media for Sf9 cells are commercially available, but they mostly still contain animal-derived components. This work aimed to develop an animal-derived component free medium for Sf9 cells. Chemically defined lipid concentrate (CDLC), yeast extract (YE), and soytone (ST) were studied for serum substitutes using a central composite design (CCD). OSF9-ADCFM contained optimum concentrations of CDLC, YE and ST at 0.5% (v/v), 11.0 g/L, and 3.0 g/L, respectively. The mean viable cell concentration of 1.71 ± 0.14 x 10⁵ cells/mL was obtained from 5 passages (P1-P5) in 25 cm² tissue culture flask. The use of both peptones after 10-kDa ultrafiltration had a significant effect on Sf9 cells growth. Maximum cell concentrations and the specific growth rates of cell adaptation in suspension culture were 1.79–2.17 x 10⁶ cells/mL and 0.025–0.027 h^− 1, respectively. The results indicated that OSF9-ADCFM could be used for Sf9 culture in both static and suspension culture. Further study would be needed to verify the effect on protein production of this in-house animal-derived component-free medium.

Animal Science

Animal-derived component-free medium

Central Composite Design

Chemically defined lipid concentrate

Spodoptera frugiperda (Sf9)

Soytone

Yeast extract

Insect cells (IC) in conjunction with the baculovirus expression vector system (BEVS), or IC-BEVS, have been widely used the production of recombinant proteins, vaccines, and a vector for gene therapy (Drugmand et al. 2012; Ikonomou et al. 2003; van Oers et al. 2015). Spodoptera frugiperda (Sf9) is a normal cell line capable of growing in a serum-free medium without any added growth factors. Sf9 cells can secrete growth factors (IGF-I) to regulate their own cell growth mechanisms (Doverskog et al. 1997). Typically, the basic formulations of the four most commonly used insect cell culture media are Grace’s, Hink’s TNM-FH, TC-100, and IPL-41. All four media are generally supplemented with 10% serum (Schlaeger 1996). In animal cell culture, sera, especially derived from fetal bovines or calves (FBS or FCS), have been supplied at 2–20% (v/v) in a basal medium to stimulate cell growth and other cellular activities (Moraes et al. 2008). The serum contains growth factors, albumin, transferrin, hormones, protease inhibitors, lipids, minerals and other essential nutrients in which facilitate the attachment and spreading of cultured cell (Hewlett 1991; Gstraunthaler 2003; Freshney 2010).

However, there are several disadvantages of using sera in cell culture media, including physiological variability, shelf life and consistency, quality control, specificity, availability, downstream processing, contamination, cost, growth inhibitors, and standardization (Freshney 2010). The collection of serum causes unneeded pain to unborn calves (van der Valk et al. 2004). In addition, the risk of contamination during the serum production process, particularly from transmissible spongiform encephalopathies (TSEs) or bovine spongiform encephalopathies (BSE), the use of animal-derived materials is not recommended for serum-free medium development (Gstraunthaler 2003).

Serum-free media are currently media that are designed to grow a specific cell type or to be used only in the absence of serum. It enables cell culture to be carried out under specific conditions with no confounding variables. Bacto peptone (Keay 1977; Sakoda and Fukusho 1998), peptone and casein hydrolysate (Saha and Sen 1989), tryptose peptone (Zhang et al. 1994a), casein peptone (Dyring et al. 1994), Primatone RL (Schlaeger 1996; Gu et al. 1997; Nyberg et al. 1999), yeastolate or yeast extract (Sung et al. 2004) have been using to combined with a basal medium to formulate animal-derived free medium. Shen et al. (2007) summarized that yeastolate fraction could promote growth activities of Sf9 ranging from 32 to 80% comparing to control group. Soy peptone, a plant-derived protein hydrolysate, has been widely used for serum-free media development of both mammalian and insect cells (Ikonomou et al. 2003; Kwon et al. 2005; Agathos 2007; Chan and Reid 2016). Apart from yeastolate and soy peptone, Chemically defined lipid concentrate is currently commercially available and designed to reduce or replace fetal bovine serum in cell culture media for a wide range of applications including CHO, hybridoma, and insect cell culture growth and maintenance, monoclonal antibody production by hybridomas, and viral expression in insect cells (Hink 1991; Goosen 1991; Schlaeger 1996; Parampalli et al. 2007; Batista et al. 2008).

Although there are many commercially available serum-free media (SFM), animal-derived component free media (ADCFM), and chemically defined media (CDM), low cost serum-free media or in-house serum-free media are still a challenging alternative to reduce the cost of insect cell cultures along with baculovirus cultures. Herein, three independent variables namely, Bacto™ Soytone (ST), Bacto™ yeast extract (YE), and chemically defined lipid concentrate (CDLC) are chosen to optimize as serum substitutes using a systematic design experiment, Central Composite Design (CCD). The growth and maintenance of Sf9 cells in the in-house animal-derived component-free medium were investigated both in static and suspension cultures. In addition, the effects of the combination of the microfiltration and ultrafiltration of both ST and YE on Sf9 cell growth were also studied.

Cell line and cell culture

The insect cell line Spodoptera frugiperda (Sf9) was purchased from the American Type Culture Collection (ATCC CRL-1711, Manassas, VA, USA). The cells were grown in a 5 mL serum-containing (SC) medium that consisted of Grace’s insect medium, 10% (v/v) FBS, 3.3 g/L lactalbumin, and 3.3 g/L yeast extract in a 25 cm² tissue culture flask at 28°C for 4 days. After that, the cells were dislodged from the flasks using a cell scraper (SPL Life Science, Gyeonggi-do, Korea). For cell counting, 10 µL of cell culture sample was diluted by adding 10 µL of a 04 % trypan blue dye solution and then loaded into a counting chamber. Total cells, live or viable cell concentrations, and death cell concentration were measured by using a Countess™ II FL automated cell counter (Thermo Fisher Scientific, USA). For subcultivation, the viable cell concentration of 4 x 10⁴ cells/cm² obtained from a previous culture was inoculated into a new 25 cm² tissue culture flask containing a 5 mL SC medium and then incubated in the same conditions as mentioned above. Cells were passaged every 4 days to a cell density of 1.2 x 10⁵–1.6 x 10⁵ cells/cm² and maintained in SC medium for 5 passages before use.

Media and supplements

Grace’s insect medium (plus, L-glutamine, minus, sodium bicarbonate, lot #1970718) was purchased from Gibco (USA). Yeast extract (YE, lot #9070604) and Soytone (ST, lot #8338681) were purchased from Becton, Dickinson and Company (USA). Chemically defined lipid concentrate (CDLC lot #1439293), MEM vitamin solution (100x, lot #982533), L-glutamine (lot #2075257), D-glucose (lot #SLCD3109), vitamin B12 (cobalamin, lot #SLBD0573V), Sodium hydrogen carbonate were purchased from Merck (Germany, lot #K40431429 943). YE and ST stock solutions were prepared by dissolving either YE or ST powder in distilled water, followed by ultrafiltration through a membrane cassette with a 10-kDa molecular weight cut-off (Pall Corporation, USA) before sterile filtration through a 0.22 µm membrane filter (Sartorius, Germany). The stock solution of methylcellulose of 1% (w/v) was made by dissolving in distilled water followed by autoclaving at 121 ^o C for 20 min.

The basal medium used in all experiments was modified from Grace’s insect medium by adding other supplements. The basal medium had the following compositions: Grace’s insect medium, 45.7 g/L; sodium hydrogen carbonate, 0.35 g/L; D-glucose, 9 g/L; vitamin B12, 0.00015 g/L; methyl cellulose, 0.1% (w/v); MEM vitamin solution (100x), 0.5% (v/v); and L-glutamine, 0.876 g/L. Pluronic F68 (0.1% w/v) was added to the basal medium for suspension culture. The basal medium was filtered sterile using a 0.22 µm membrane filter before use. For the experiments, the three independent variables to be studied (CDLC, YE, ST) were separately added to the basal medium according to the specified concentration in the design of experiments.

Experimental design

The software Minitab R16 (Minitab Inc., USA) was used for designing the experiments and the statistical analysis of the data. Response surface methodology (RSM) was used to determine the influence of the supplements (CDLC, YE, ST) on the response (viable cell yields). A circumscribed central composite design (CCD) for three independent variables at five levels each was constructed as shown in Table 1. The independent variable coding regions were − α (− 1.682, lowest level), − 1, 0 (middle level), + 1, and + α (+ 1.682, highest level). The experiments were simultaneously done in triplicate for 4 days. The treatment combinations of the three independent variables on the responses (Y) were presented in Table 2.

Table 1

Actual factor levels corresponding to coded factor levels
Variables	Symbols (Unit)	Coded factor levels
Variables	Symbols (Unit)	-1.682	-1	0	1	+ 1.682
CDLC	X1 (%)	0.33	0.50	0.75	1.00	1.17
YE	X2 (g/L)	0.27	3.00	7.00	11.00	13.73
ST	X3 (g/L)	0.27	3.00	7.00	11.00	13.73

Table 2

The treatment combinations of variables in coded values, the values of responses (Y) obtained from the experiments and prediction
Run	Factors			Viable cell yield (cells/mL)
Run	X1	X2	X3	Experimental value	Predicted value
1	-1	-1	-1	9.33E + 4	1.04E + 5
2	+ 1	-1	-1	2.66E + 4	4.33E + 4
3	-1	+ 1	-1	2.06E + 5	1.98E + 5
4	+ 1	+ 1	-1	4.00E + 4	4.37E + 4
5	-1	-1	+ 1	1.06E + 5	9.88E + 4
6	+ 1	-1	+ 1	3.33E + 4	3.78E + 4
7	-1	+ 1	+ 1	1.73E + 5	1.52E + 5
8	+ 1	+ 1	+ 1	1.33E + 4	-1.83E + 3
9	-1.682	0	0	1.80E + 5	1.93E + 5
10	+ 1.682	0	0	2.00E + 4	1.26E + 4
11	0	-1.682	0	4.00E + 4	2.34E + 4
12	0	+ 1.682	0	4.66E + 4	6.89E + 4
13	0	0	-1.682	1.40E + 5	1.24E + 5
14	0	0	+ 1.682	6.00E + 4	8.13E + 4
15	0	0	0	7.33E + 4	7.30E + 4
16	0	0	0	6.66E + 4	7.30E + 4
17	0	0	0	8.00E + 4	7.30E + 4
Note. Coded variable levels are − α = −1.682, +α = +1.682, − 1, 0, + 1; response (Y) = the average value of viable cell concentration obtained from each run (n = 3) on day 4 of culturing. Symbols X1, X2, and X3 are CDLC, YE, and ST, respectively.

Cell culture

The starter cells obtained were scrapped and washed three times in phosphate buffer saline (Ca²⁺, Mg²⁺-free PBS, pH 7.4) by centrifugation at 110 x g for 10 min. After that, the cell pellets were resuspended in PBS obtaining the cell concentration of 10⁶-10⁷ cells/mL. The initial cell density of 4 x 10⁴ cells/cm² was placed in each 6-well plate containing 3 mL of a tested medium, then the plates were incubated at 28 ^o C for 4 days. The experiment for each tested medium was carried out in triplicate for 4 days. A small volume of suspended Sf9 cells was taken into 1.5 ml microtube, then the concentration of viable cells was counted by using a Countess™ II FL automated cell counter as mentioned above.

Statistical analysis

The responses (Y) were first plotted according to the Anderson–Darling statistic in Minitab software (Minitab Inc., USA) for statistical analysis. The probability plot of the responses (as residues) on a specified day of the cultures is constructed. If the p-value of the Anderson–Darling statistic ≥ 0.05, indicating the normal distribution of the data. Next, the regression analysis was performed using the analyze response surface design function, and terms including linear, linear + squares, linear + interactions, and full quadratic were selected. The value of the coefficient of determination (R²) was used to evaluate the sufficiency of the model. The selected model depended on the selected terms that always gave a p-value ≤ 0.05. The reduced model based on the condition t-test in the function was evaluated using the R² value. A second-order or higher-order polynomial regression model was fitted as described in Eq. (1) using the data presented in Table 2.

$$\text{Y}\text{ }\text{=}\text{ }{\text{b}}_{\text{0}}\text{ }\text{+}\sum _{\text{i=}}^{\text{k}}{{\text{b}}_{\text{i }}\text{X}}_{\text{i }}\text{+}\sum _{\text{j=}}^{\text{k}}{\text{b}}_{\text{i}\text{i}}\text{X}{\text{i}}^{\text{2}}\text{ }\text{+}\sum \sum {\text{b}}_{\text{ij}}{\text{X}}_{\text{ij }}\text{+}\text{ }\text{ε}$$

where

Y = Viable cell yield, b₀ = Intercept value

X = Factors (X1 = CDLC, X2 = YE, X3 = ST)

b _z = Regression coefficient (z = i, ii or ij, where i < j)

ε = Residual error, k = 1, 2, 3…

The p-value of the lack-of-fit for the selected model should be ≥ 0.05. A pair sample Student’s t-test was used to determine the significance of differences among the groups in the experiments.

Verification study

The optimum concentrations of the three independents in the basal medium (named “OSF9-ADCFM”) were validated. Cell inoculums were cultivated in an SC medium. The initial viable cell density of 4 x 10⁴ cells/cm² was used to inoculate to a 25 cm² tissue culture flask, and then a volume of the culture medium was added up to 5 mL. The cultured flasks were carried out in triplicate. The cells in the cultured flasks were incubated at 28°C for 3–5 h until cells were attached to the flasks. After cell attachment, the spent medium was carefully removed and gently replaced with 5 mL OSF9-ADCFM. The cultured flasks were continuously incubated at 28°C for 4 days. Subsequently, the cells were mildly harvested using a cell scraper. The concentration of viable cells was measured as mentioned above. The cells obtained from this first optimized culture were named passage 0 (P0). To subculture in the next passage, the P0 cell were centrifuged at 110 x g for 5 min, and then the cell pellets were resuspended in OSF9-ADCFM at a specified volume. After that, the initial viable cell density used for the experiments was equal to that used above, and 5 mL OSF9-ADCFM was used instead of SC media. The efficiency of OSF9-ADCFM in maintaining and supporting the growth of cultured cells was recorded as the viable cell yield, and the cultured cells were continuously subcultured for 5 passages.

Study of the optimum cell density in the static culture

The optimum cell density was evaluated using various initial cell densities placed in a 25 cm² tissue culture flask containing 5 mL of OSF9-ADCFM: 2 x 10⁵, 3 x 10⁵, 4 x 10⁵, and 5 x 10⁵ cells/mL (equal to 4 x 10⁴, 6 x 10⁴, 8 x 10⁴ and 1 x 10⁵ cells/cm², respectively). Each initial cell density was carried out in triplicate. All flasks were incubated at 28°C for 4 days. The viable cells were dislodged from the flasks using a cell scraper and then counted as previously mentioned.

The combined effect of microfiltration and ultrafiltration of YE and ST on Sf9 cell growth

Stock solutions of YE and ST were made by dissolving either YE or ST powder in distilled water. For the experiments, YE and ST were filtered through a 0.22 µm membrane filter (named “Fraction #1ST” and “Fraction#1YE”); then the Fraction #1 was ultrafiltered through a membrane cassette (Pall Corporation, USA) with a molecular weight cut-off of 10 kDa (named “Fraction #2ST” and “Fraction#2YE”). At the lower cut-off, Fraction #2 was ultrafiltered through a membrane cassette with a molecular weight cut-off of 3 kDa (named “Fraction #3ST” and “Fraction#3YE”). The filtered products obtained from Fraction #1, Fraction #2, and Fraction #3 were kept at 2–8°C before use. For the experiments, the combinations of Fractions obtained from both ST and YE (RUN1-9) were shown in Table 3.

Table 3

Experimental design of the combined effect of various filtrated ST and YE
Run no.	Combination effect
1	Fraction #1ST + Fraction #1YE
2	Fraction #1ST + Fraction #3YE
3	Fraction #1ST + Fraction #2YE
4	Fraction #3ST + Fraction #1YE
5	Fraction #3ST + Fraction #3YE
6	Fraction #3ST + Fraction #2YE
7	Fraction #2ST + Fraction #1YE
8	Fraction #2ST + Fraction #3YE
9	Fraction #2ST + Fraction #2YE

The final concentration of YE and ST were 11 g/L, and 3 g/L, respectively, while the final concentration of CDLC was 0.5% (v/v). Regarding the final concentrations of both YE and ST in terms of protein content, thus the total protein concentrations of YE and ST were measured using NanoDrop (ND-1000) Spectrophotometer (Thermo Scientific, USA). The starter cells were grown in OSF9-ADCFM in 175 cm² tissue culture flasks, and then the cells were scraped and washed three times in PBS. Cells with a density of 4 x 10⁴ cells/cm² were placed in 25 cm² tissue culture flasks containing 5 mL of each fraction combination and carried out in triplicate. All flasks were incubated at 28°C for 4 days. Subsequently, the cells in all flasks were scraped and mixed well before a small volume of cell sample from each tested medium was taken to be counted the viable cell concentration using the method as mentioned above. Osmolality of medium was measured using Cryoscopic Osmometer (OSMOMAT^® 030, Gonotec Gesellschaft für Meẞ-und Regeltechnik mbH, Germany)

Adaptation of Sf9 cells to grow in suspension

Suspension cultures of Sf9 cell growth in OSF9-ADCFM was investigated. The initial viable cell concentration of 4 x 10⁴ cells/cm² was grown in a 175 cm² tissue culture flask containing 30 mL OSF9-ADCFM medium at 28°C for 4 days. Then, the viable cell density of 3 x 10⁵ cells/mL was inoculated in 250 mL spinner flasks containing 50 mL of medium. The spinner flasks were stirred at 50 rpm and 28°C. Cell culture samples were taken daily to count the viable cells according to the method described above. The subcultivation of cells was carried out, when the concentration of viable cells reached 1–2 x 10⁶ cells/mL, by taking the viable cell density of 3 x 10⁵ cells/mL and placing in a new spinner flask containing 50 mL of fresh medium. The growth of the cells was continuously cultured for 5–7 days. The morphology of cultured Sf9 cells was photographed. The evaluation of cell growth in the spinner flasks was carried out separately for two batches.

Regression analysis of the selected model

The analysis of variance (ANOVA) for the data of viable cell yield on day 4 of culturing indicated that this model was significantly effective with the p-values ≤ 0.05. It was found that X1 (0.000), X2 (0.047), and X1 x X2 (0.016) had p-values ≤ 0.05, indicating that they all were significantly effective on viable cell yield (Table 4). Based on the t-values obtained, CDLC (X1:-9.568) had the greatest negative effect on viable cell concentrations. YE (X2: 2.400) had a slightly positive effect on viable cell concentrations, while ST (X3: -2.270) had a slightly negative effect. The pairwise interaction between CDLC and YE (X1 x X2: -3.172) had the greatest negative effect on viable cell concentrations, while all the squares (X1 x X1: 1.703, X2 x X2: -1.529, X3 x X3: 1.703) had a slight effect on viable cell concentrations (Table 5). The coefficient of determination or R² value for the full quadratic model was 94.70%, while R² (adjusted) was also high at a value of 87.88%, indicating that the correlation of all variables and the response could be described by the model. Moreover, it was also found that the p-value of lack-of-fit was relatively high at a value of 0.072 (Table 4), which indicates that the model was adequate for estimating the response.

Table 4

Analysis of variance (ANOVA) for the data of cell concentrations on day 4 of culturing
Source	DF	Sums of Squares	Mean Squares	F	p-values
Regression	9	5.41E + 10	6.01E + 9	13.89	0.001
Linear	3	4.43E + 10	1 47E + 10	34.15	0.000
X₁	1	3.96E + 10	3.96E + 10	91.55	0.000
X₂	1	2.49E + 9	2.49E + 9	5.76	0.047
X₃	1	2.23E + 9	2.23E + 9	5.15	0.057
Square	3	4.60E + 9	1.53E + 9	3.54	0.076
X₁X₁	1	1.38E + 9	1.25E + 9	2.90	0.132
X₂X₂	1	1.95E + 9	1.01E + 9	2.34	0.170
X₃X₃	1	1.25E + 9	1.25E + 9	2.90	0.132
Interaction	3	5.15E + 9	1.718E + 9	3.97	0.061
X₁X₂	1	4.35E + 9	4.35E + 9	10.06	0.016
X₁X₃	1	0	0	0.00	1.000
X₂X₃	1	8.00E + 8	8.00E + 8	1.85	0.216
Residual Error	7	3.03E + 9	4.32E + 8
Lack-of-Fit	5	2.94E + 9	5.88E + 8	13.24	0.072
Pure Error	2	8.88E + 7	4.44E + 7
Total	16	5.71E + 10

Table 5

Estimated regression coefficients for the data of cell concentrations on day 4 of culturing
Term	Coefficients	SE coefficients	T	p-values
Constant	73006	11990	6.089	0.000
X₁	-53874	5631	-9.568	0.000
X₂	13513	5631	2.400	0.047
X₃	-12781	5631	-2.270	0.057
X₁X₁	10557	6197	1.703	0.132
X₂X₂	-9478	6197	-1.529	0.170
X₃X₃	10557	6197	1.703	0.132
X₁X₂	-23333	7357	-3.172	0.016
X₁X₃	-0.1	7357	-0.000	1.000
X₂X₃	-10000	7357	-1.359	0.216
S = 20807.6 PRESS = 22595653257
R² = 94.70% R² (predicted) = 60.46%
R² (adjusted) = 87.88%

To construct the model, all coefficients and symbols of all variables in the forms of linear, squares, and interactions were arranged in a series as presented below. The full quadratic model in the form of the mathematical equation was shown in Eq. (2).

Y = 73006–53874X1 + 13513X2–12781X3 + 10557 (X1 x X1) − 9478 (X2 x X2) + 10557 (X3 x X3) − 23333 (X1 x X2) − 0.1 (X1xX3) − 10000 (X2 x X3) (2)

The optimum points of three independent variables derived from Response Optimizer function of software Minitab R16 (Minitab Inc., USA) were 0.5% (v/v) CDLC (X1), 11 g/L YE (X2), and 3 g/L ST (X3). Generally, the cell concentration estimated according to the empirical mathematical model was 1.98 x 10⁵ cells/mL.

Three-dimensional (3D) response surface plots were created by the Design Expert 13.0 software to examine the optimum concentration for all possible combinations (Fig. 1). The interaction between CDLC + YE in this range showed a strong effect on Sf9 growth (p-value ≤ 0.05). Increasing CDLC concentrations resulting in declining of viable cell while YE promoted a better growth (p-value ≤ 0.05). The interaction between CDLC + ST and ST + YE in the tested range indicated that they slightly affected on Sf9 growth (p-value ≥ 0.05). ST and YE have been used extensively as a medium substitute as a source of vitamins, amino acids, peptides and carbohydrates in serum-free medium to improve cell density and productivity (Posung et al. 2021). The result from Spearman et al. revealed that soy hydrolysate stimulated growth of CHO cells while yeast hydrolysate caused reduction in their growth (Spearman et al. 2014). On the other hand, yeastolate fraction could promote growth activities of Sf9 ranging from 32 to 80% compared to control group. (Shen et al. 2007). Andreassen et al. concluded that YE alone could restore cell growth and replace FBS during bovine muscle cell cultivation in serum-free media (Andreassen et al. 2020). Anyhow, which components of yeast extract that are responsible for the growth-promoting effects could not be fully described (Mosser et al. 2015). Low dose (1 g/L) of ST could stimulate growth of Keratinocyte cell up to 201% whereas higher concentrations resulting in growth inhibition (Lee et al. 2008). Lee et al. also suggested that adherent cell cultures may need lower protein sources than suspension culture like CHO cell (Lee et al. 2008). In addition, amino acid utilization in cells results in ammonia by-product accumulation. Therefore, high amino acids can cause cell growth inhibition by high ammonia content in the medium (Zhang et al. 1994b). In this circumstance, YE could support better Sf9 growth than ST. It might be because the protein compositions of both hydrolysates are different. Most of protein in YE are less than 250 daltons (62%) while ST had a small unit protein only 40% of total (Thermofisher 2019). Kudełka et al. pointed that most of proteins in ST are globulin which is a high molecular weight protein and also contained protease inhibitor (Kudełka et al. 2021) that may not be suitable for Sf9 growth. The analyzed model revealed that increasing ST from 3 g/L to 11 g/L led to lower cell yield (data not shown). This inhibition probably due to some components in ST. Lipids and related components are served in biological function: cell membrane components, nutrient storage and transport, and signal transduction (Yao and Asayama 2017). Formulation of serum-free medium are necessary to include lipids at a concentration range of 10–100 µg/L whereas insect cell need much higher at 1,000 µg/L (Shen et al. 2004). Cholesterol in CDLC is an important constituent in serum-free media for Sf9 because they cannot synthesize by themselves (Mitsuhashi 1989). The analyzed model revealed that CDLC at 0.5% (v/v) gave the highest viable cell yield (data not shown) while increment of concentration in formulated medium cause the decline of cell yield. It was probably due to the higher hydrophobic of CDLC that directly contact to cell without protection from FBS could reduce Sf9 growth (Batista et al. 2008).

Verification study

The verification study was performed using the basal medium supplemented with three independent variables at the optimum concentrations. The medium containing 0.5% (v/v) CDLC, 11 g/L YE and 3 g/L ST was named OSF9-ADCFM. The viable cell concentration of Sf9 cells grown in OSF9-ADCFM during a continuous subcultivation for 5 passages (P1-P5) was presented in Fig. 2. In the first passage P0, Sf9 inoculum was first transferred from serum-containing medium which residual serum proteins in inoculum might stimulate cell growth after replacing with OSF9-ADCFM. Maximum viable cell concentration of 1.91±0.15–1.93±0.15 x 10⁵ cells/mL (P2-P4) was obtained with relative cell number greater than 107% compared to P0 and thereafter the viable cell concentration rapidly decreased (1.20±0.10 x 10⁵ cells/mL) with relative cell number of 67%. However, even the number of viable cells could not increase at the last passage P5, but the percent of cell viability was still high enough (97%±2%) to be used for a long-term subcultivation. Overall, the average viable cell concentration of 1.71 ± 0.14 x 10⁵ cells/mL and the average percent of cell viability of 96.8% ± 1.00% were obtained from 5 passages (P1-P5). The obtained average viable cell yields under optimal condition were in the accepted range of 1.68 x 10⁵ – 2.27 x 10⁵ cells/mL as calculated from the empirical mathematical model indicating the suitability of this model (Posung et al. 2021). The osmolality of OSF9-ADCFM was 511 ± 3 mOsm/kg (n = 3) which was much greater than the commercial serum free media at 345–359 mOsm/kg (Doverskog et al. 2000). However, the reported values of medium osmolality for insect cells could vary from 250 to 500 mOsm/kg (Zhang et al. 1994a), while they also reported that the growth rate of Silkworm (Bombyx mori, Bm5) was maximal at a medium osmolality of about 370 mOsm/kg, and more than 90% of the maximum growth rate was achieved with osmolality between 350 and 385 mOsm/kg (Zhang et al. 1994a). For the cultivation of Trichoplusia ni BTI-TN-5B1-4 (High-Five™), it was found that the shift up in osmotic pressure from 350 to 500 mOsm/kg caused no significant differences in cell proliferation rate during exponential growth phase (Olejnik et al. 2003). Batista et al. (2005) found that Grace’s medium supplemented with 2.7 g/L glucose, 8 g/L YE, 0.1% (w/v) Pluronic F-68, 1% (w/v) milk whey ultrafiltrate (MWU), and 3% (v/v) FBS had the osmolality up to 438 mOsm/kg but it could stimulate Sf9 cell growth by about 5-fold (4.7 x 10⁶ cells/mL) when compared to Grace’s contaiing 10 % (v/v) FBS (9.5 x 10⁵ cells/mL). In addition, Batista et al. (2008) also found that supplemented IPL-41 containing 6 g/L yeastolate ultrafiltrate, 10 g/L glucose, 3.5 g/L glutamine, 0.5 g/L fructose, 2 g/L lactose, 0.6 g/L tyrosine, 1.48 g/L methionine and 1 % (v/v) lipid emulsion had the osmolality up to 460 mOsm/kg but it could still support growth of Drosophila melanogaster S2 (S2AcGPV) up to the maximal cell concentration of 19 x 10⁶ cells/mL. We noticed that a modified Grace’s insect medium used as such a basal medium had the osmolalilty of 413 ± 2 mOsm/kg (n = 3), it was possible that the addition of YE and ST at 11 g/L and 3 g/L could significantly increase the osmolality of OSF9-ADCFM. We assumed that osmolality did not affect Sf9 growth, if so, they would not grow in even the first passage. Lower cell yield in serum-free medium is common because of inadequate nutrients compared to the medium containing serum.

Optimum cell density in the static culture

The optimum initial cell density in static culture was tested in the range of 2–5 x 10⁵ cells/mL in OSF9-ADCFM. The use of a low cell concentration (2 x 10⁵ cells/mL) could not achieve a high cell concentration at the end of 5 days culturing (Fig. 3A). However, when the initial cell concentration was higher than 2 x 10⁵ cells/mL, the final cell concentration was noticeably higher. Initial cell concentration of 3–5 x 10⁵ cells/mL gave almost the same level of final cell concentration. Nevertheless, an initial cell concentration of 3 x 10⁵ cells/mL could give the maximum multiplication ratio of 6.69 ± 0.15, while those of the initial cell concentrations of 2 x 10⁵, 4 x 10⁵, and 5 x 10⁵ cells/mL were 1.34 ± 0.07, 5.32 ± 0.17, and 4.05 ± 0.09, respectively (Fig. 3B). Parizi et al. concluded that the optimum initial cell density of BHK1 cells in suspension culture was 3x10⁵ cells/mL with the highest multiple ratio of 3.7 (Parizi et al. 2017). High cell density engendered the competition for obtaining the limited nutrients and also influenced the pH of media. Meanwhile, some signal produced from cells could stimulate the growth of others (Parizi et al. 2017). Spens suggested that increasing amount of serum or specific growth factors in medium could decrease the critical initial cell density (cID) (Spens 2006). Moreover, inoculation of initial cells below cID, viable cell density would be declined before they started to proliferate (Spens 2006). The optimum initial cell density would be different between cell lines and medium used. Hence, the optimum initial cell concentration for Sf9 growth in OSF9-ADCFM was 3 x 10⁵ cells/mL.

Effect of microfiltration and ultrafiltration components of YE and ST

The effect of protein fraction on Sf9 growth were done by microfiltration and ultrafiltration of YE and ST, then each fraction was combined as shown in Table 3. The fraction #1, #2, and #3 had the protein molecular weight between < 250 Da and > 10 kDa, < 10 kDa, and < 3 kDa, respectively. The results exhibited that the viable cell concentrations obtained from the combinations of microfiltration and ultrafiltration of YE and ST at Run 6, Run 8, and Run 9 were respectively 6.27±0.07 x 10⁵, 5.98±0.13 x 10⁵, and 7.17±0.08 x 10⁵ cells/mL which were relatively higher than those of other combinations as shown in Fig. 4. It was noticed that only some combinations of microfiltration and ultrafiltration of both YE and ST could stimulate growth of Sf9 cells, namely RUN6: Fraction #3ST + Fraction #2YE, RUN8: Fraction #2ST + Fraction #3YE, and RUN9: Fraction #2ST + Fraction #2YE. We observed that the combination of high molecular weight of YE and ST (RUN 1) could not support good growth of Sf9 compared to others. Moreover, RUN 5 that contained #3ST and #3YE showed the lowest viable cell concentration. Fraction #3 contained all free amino acid, which has molecular weight in the range of 75–204 daltons, and some oligopeptides (Mustățea et al. 2019). However, some carbohydrates may be removed from this fraction. Therefore, it might not be enough nutrients for Sf9 to grow in this combination. RUN 1, 2 and 4 in which showed similar viable cell concentrations, they all contained fraction #1 and lacked fraction #2. Combination in RUN 3 and 7, exhibited better growth than RUN 1, 2 and 4, contained fraction #1 and fraction #2. While RUN 6, 8 and 9 all contained fraction #2. We believed that high molecular weight of YE and ST in fraction #1 may contain some inhibition factor for Sf9 whereas 10 kDa in fraction #2 of both YE and ST might be suitable for its growth. However, the scientific reason of such a synergistic effect is still required to be further explained. A paired samples Student’s t-test indicated that the final cell concentration obtained from RUN9 was significantly different from the other two. In fact, ST and YE supplemented in RUN9 were the same composition as in OSF9-ADCFM. Mendonça et al. (2007) found that low molecular weight (LMW) of YE (less than 30 kDa) had a positive effect on growth of Sf9 cells better than high molecular weight (HMW). Shen et al. (2007) used sequential ethanol precipitation of yeastolate ultrafiltrate (YUF1) combined with other YUFs to culture Sf9 cells and they showed synergistic effect to enhance Sf9 growth. Also, Chou (2013) noticed that the low molecular weight of yeastolate at less than 10-14.2 kDa could fully support cell growth of IPLB-Sf-21AE (Sf21). Our results revealed that ultrafiltration (10 kDa cut-off) of YE and ST could enhance Sf9 growth that corresponds to reports from the previous study. However, there is not much enough information on how its synergistic effect of ST on insect cells. Nevertheless, integration of an ultrafiltration step in media preparation can eliminate hydrolysate lot variability and ensure reproducible good growth of insect cell lines (Schlaegae 1996; Ikonomou et al. 2003).

Adaptation of Sf9 cells to grow in suspension

The adaptation of Sf9 cells to grow in suspension was carried out in 250 mL spinner flasks containing OSF9-ADCFM with 3 x 10⁵ cells/mL initial cell concentration. The growth of Sf9 cells was investigated separately in two batches as shown in Fig. 5. The maximum viable cell concentrations obtained from batches no. 1 and no.2 were 1.79 x 10⁶ and 2.17 x 10⁶ cells/mL, respectively. The maximum multiplication ratio of batch no. 1 and no. 2 were 4.96 on day 5 and 6.23 on day 4, respectively. Hensler et al. (1994) believed that the lag phase is population density-associated. They suggested that seeding densities should be higher than 6 x 10⁵ cells/mL to overcome the long culture lag of Sf9 growth in serum-free medium (Hensler et al. 1994). Since the variation in cell numbers was observed during the culturing of both batch no.1 and batch no. 2, the kinetic parameters (the number of generations of cell growth, doubling time, and specific growth rate) were calculated as follows: batch no.1, were 1.72, 28 h, and 0.025 h^− 1, respectively while batch no. 2, were 1.90, 26 h, and 0.027 h^− 1, respectively. Our results indicated that the calculated kinetic parameters were not statistically different, indicating a similar growth pattern of Sf9 cells in OSF9-ADCFM. Doubling time and specific growth rate of Sf9 culture in OSF9-ADCFM were close to Sf9 culture in EX-CELL 420 with 26 h and 0.026 h^–1, respectively (Aytekin et al. 2016). We noticed that cells became aggregates in batch no. 1 and the average diameter of Sf9 cells was 12.8 µm (Fig. 6). Dextran sulfate (0.25 g/L) was added in batch no. 2. to minimize cell clumping. Dextran sulfate successfully reduced cell aggregation, which might be the reason for the higher countable cell number in batch no. 2. Based on the results, Sf9 cells could be adapted to grow and maintained in OSF9-ADCFM.

An animal-derived component-free medium development by CCD indicated that CDLC exhibited a strong negative effect, YE had slightly positive effect and ST had slightly negative effect on viable cell yield. CDLC, YE, and combination of CDLC and YE revealed significantly effective on viable cell yield. The optimum points of CDLC, YE and ST of OSF9-ADCFM containing 0.5% (v/v) CDLC, 11 g/L YE and 3 g/L ST. The verification study of Sf9 in OSF9-ADCFM continuously in 5 passages (P1-P5) had the average viable cell concentration of 1.71 ± 0.14 x 10⁵ cells/mL which were in the range of 1.68 x 10⁵ – 2.27 x 10⁵ cells/mL as calculated from the empirical mathematical model indicating the suitability of this model. The optimum initial cell concentration was 3 x 10⁵ cells/mL. Both peptones after 10-kDa ultrafiltration significantly affect Sf9 growth. Two batches of suspension culture in OSF9-ADCFM of Sf9 cell exhibited that it could adapt to grow in suspension culture with doubling time and specific growth rate of 28 h and 0.025 h^− 1 in batch no. 1 and 26 h and 0.027 h^− 1 in batch no. 2. The maximum viable cell concentrations obtained from batch no. 1 and no.2 were 1.79 x 10⁶ and 2.17 x 10⁶ cells/mL, respectively. The results indicated that in-house OSF9-ADCFM could be used for Sf9 culture in both static and suspension culture. The production of recombinant proteins by the BEVS will be further investigated.

Acknowledgements we would like to express our gratitude to Medical Life Sciences Institute, Department of Medical Sciences, Ministry of Public Health and Division of Biology, Faculty of Science and Technology, Rajamangala University of Technology Thanyaburi for supporting our work. Our sincere thanks also go to the staff at the Innovation and Medical Biotechnology Center (iMBC), Medical Life Sciences Institute, Department of Medical Sciences, Ministry of Public Health for their support. Finally, our special thanks to Assist. Prof. Dr. Kanokwan Poomputsa for her valuable comments of this article.

Authors' contributions Conceptualization: MP, JT, PD; methodology: SS, AA, JT, MP, ST; formal analysis: SS, AA, MP; resources: PD, MP, ST; writing-original draft: SS; writing-review and editing: MP, JT; supervision: AA, JT, MP.

Conflicts of interest The authors declare that they have no conflict of interest to disclose.

Agathos SN (2007) Development of serum-free media for Lepidopteran insect cell lines. Murhammer DW (ed), Baculovirus and Insect Cell Expression Protocols, Method in Molecular Biology. Vol 388, Humanna Press Inc., Totowa, NJ
Andreassen RC, Pedersen ME, Kristoffersen KA, Beate Rønning S (2020) Screening of by-products from the food industry as growth promoting agents in serum-free media for skeletal muscle cell culture. Food & Function 11: 2477–2488
Aytekin Ö, Gurhan SİD, Ohura K, Imai T, Öngen G (2016) Production of recombinant human dipeptidyl peptidase IV from Sf9 cells in microbial fermenters. Turkish Journal of Biology 40: 217–228
Batista FR, Pereira CA, Mendonça RZ, Moraes AM (2005) Enhancement of Sf9 cells and baculovirus production employing Graces’s medium supplemented with milk whey ultrafiltrate. Cytotechnology 49(1): 1–9
Batista FR, Pereira CA, Mendonça RZ, Moraes AM (2008) Formulation of a protein-free medium based on IPL-41 for the sustained growth of Drosophila melanogaster S2 cells. Cytotechnology 57(1): 11–22
Chan LC, Reid S (2016) Development of serum-free media for Lepidopteran insect cell lines. Methods Mol Biol 1350:161–196
Chou CC (2013) Identification of bioactive yeastolate fractions responsible for insect cell growth and baculovirus production. J Biochem Tech 4 (3): 611–615
Doverskog M, Ljunggren J, öhman L, Häggström L (1997) Physiology of cultured animal cells. J. Biotechnol 59: 103–115
Doverskog M, Bertram E, Ljunggren J, Ohman L, Sennerstam R, Häggström L (2000) Cell Cycle Progression in Serum-Free Cultures of Sf9 Insect Cells: Modulation by Conditioned Medium Factors and Implications for Proliferation and Productivity. Biotechnol Prog16: 837–846
Drugmand JC, Schneider YJ, Agathos SN (2012) Insect cells as factories for biomanufacturing. Biotechnol Adv 30: 1140–1157.
Dyring C, Hansen HA, Emborg C (1994) Observations on the influence of glutamine, asparagine and peptone on growth and t-PA production of Chinese hamster ovary (CHO) cells. Cytotechnology 16: 37–42
Freshney IR (2010) Culture of animal cells: a manual of basic technique and specialized applications (6th ed). John Wiley & Sons, Inc., New York USA, pp.115–121
Goosen MFA (1991) Large-scale insect cell culture: methods, applications and products. Curr Opin Biotechnol 2: 365–369
Gstraunthaler G (2003) Alternatives to the Use of Fetal Bovine Serum: Serum-free Cell Culture. ALTEX, 20(4): 275–281
Gu X, Xie L, Harmon BJ, Wang DIC (1997) Influence of Primatone RL Supplementation on Sialylation of Recombinant Human Interferon-γ Produced by Chinese Hamster Ovary Cell Culture Using Serum-Free media. Biotechnol Bioeng 56: 353–360
Hensler W, Singh V, Agathos SN (1994) Sf9 insect cell growth and β-galactosidase production in serum and serum-free media. Ann N Y Acad Sci 745:149–166
Hewlett G (1991) Strategies for optimising serum-free medium. Cytotechnology 5: 3–14
Hink WF (1991) A serum-free medium for the cultivation of insect cells and production of recombinant proteins. In Vitro Cell Dev Biol 27A: 397–401
Ikonomou L, Schneider YJ, Agathos SN (2003) Insect cell culture for industrial production of recombinant proteins. Appl Microbiol Biotechnol 62: 1–20
Keay L (1977) The growth of L-cells and Vero cells on an autoclavable MEM-peptone medium. Biotechnol Bioeng 19: 399–411
Kwon MS, Dojima T, Park EY (2005) Use of plant-derived hydrolysates for enhancing growth of Bombyx mori (silkworm) insect cells in suspension culture. Biotechnol Appl Biochem 42: 1–7
Kudełka W, Kowalska M, Popis M (2021) Quality of Soybean Products in Terms of Essential Amino Acids Composition. Molecules. 26(16): 5071
Lee YK, Kim SY, Kim KH, Chun BH, Lee KH, Oh DJ, Chung N (2008) Use of soybean protein hydrolysates for promoting proliferation of human keratinocytes in serum-free medium. Biotechnol Lett 30(11): 1931–1936
Mendonça, RZ, de Oliveira EC, Pereira CA, Lebrum I (2007) Effect of bioactive peptides isolated from yeastolate, lactalbumin and NZCase in the insect cell growth. Bioprocess Biosyst Eng 30:157–164
Mitsuhashi J (1989) Invertebrate cell system applications. In: Mitsuhashi J (ed). CRC Press, pp 3–20
Moraes ÂM, Mendonça RZ Suazo CAT (2008) Culture media for animal cells. In: Castilho LR, Moraes ÂM, Augusto EFP, Butler M (eds) Animal Cell Technology: From Biopharmaceuticals to Gene Therapy. Taylor & Francis Group, New York USA, pp 111–128
Mosser M, Kapel R, Chevalot I, Olmos E, Marc I, Marc A, Oriol E (2015) Fractionation of yeast extract by nanofiltration process to assess key compounds involved in CHO cell culture improvement. Biotechnol Prog 31: 875–882
Mustățea G, Ungureanu EL, Iorga E (2019) Protein acidic hydrolysis for amino acids analysis in food - progress over time: a short review. Journal of Hygienic Engineering and Design 26: 81–87
Nyberg GB, Balcarcel RR, Follstad BD, Stephanopoulos G, Wang DIC (1999) Metabolism of peptide amino acids by Chinese hamster ovary cells grown in a complex medium. Biotechnol Bioeng 62: 324–335
Olejnik A, Grajek W, Marecik R (2003) Effect of hyperosmolarity on recombinant protein productivity in baculovirus expression system. J Biotechnol 102: 291–300
Parampalli A, Eskridge K, Smith L, Meagher MM, Mowry MC, Subramanian A (2007) Developement of serum-free media in CHO-DG44 cells using a central composite statistical design. Cytotechnology 54: 57–68
Parizi SS, Ardjmand M, Mahravani H, Azimi M (2017) Effects of seed, serum and media composition on growth
and proliferation of BHK cells in suspension culture in a stirred bioreactor. Bulgarian Chemical Communications 49: 297–307
Posung M, Promkhatkaew D, Borg J, Tongta A (2021) Development of a modified serum-free medium for Vero cell cultures: effects of protein hydrolysates, L-glutamine and SITE liquid media supplement on cell growth. Cytotechnology 73: 683–695
Saha SN, Sen AK (1989) Studies on the development of a medium with peptone and casein hydrolysate for the production of foot-and-mouth disease vaccine in BHK-21 cells. Vaccine 7: 357–363
Sakoda Y, Fukusho A (1998) Establishment and characterization of a porcine kidney cell line, FS-L3, which forms unique multicellular domes in serum-free culture. In Vitro Cell Dev Biol 34: 53–57
Schlaeger EJ (1996) Medium design for insect cell culture. Cytotechnology 20: 57–70
Shen CF, Hawari J, Kamen A (2004) Micro-quantitation of lipids in serum-free cell culture media: a critical aspect is the minimization of interference from medium components and chemical reagents. J Chromatography B 810(1): 119–127
Shen CF, Kiyota T, Jardin B, Konishi Y, Kamen A (2007) Characterization of yeastolate fractions that promote insect cell growth and recombinant protein production. Cytotechnology 54: 25–34
Spearman M, Lodewyks C, Richmond M, Butler M (2014) The bioactivity and fractionation of peptide hydrolysates in cultures of CHO cells. Biotechnol Prog 30(3), 584–593.
Spens E (2006) Development of a protein-free fed-batch process for NS0 cells: Studies on regulation of proliferation. School of Biotechnology, Department of Bioprocess Technology, Royal Institute of Technology (KTH), SE-106 91 Stockholm, Sweden. ISBN 91-7178-466-7
Sung YH, Lim SW, Chung JY, Lee GM (2004) Yeast hydrolysate as a low-cost additive to serum-free medium for the production of human thrombopoietin in suspension cultures of Chinese hamster ovary cells. Appl Microbiol Biotechnol 63:527–536
Thermofisher scientific (2019) Technical guide to peptones, supplements, and feeds Enhancing performance of mammalian and microbial bioprocesses. Available online https://assets.thermofisher.com/TFS-Assets/BPD/brochures/peptones-supplements-feeds-technical-reference-guide.pdf
van der Valk J, Mellor D, Brands R, Fischer R, Gruber F, Gstraunthaler G, Hellebrekers L, Hyllner J, Jonker FH, Prieto P, Thalen M, Baumans V (2004) The humane collection of fetal bovine serum and possibilities for serum-free cell and tissue culture. Toxical in Vitro 18: 1–12
van Oers MM, Pijlman GP, Vlak JM (2015) Thirty years of baculovirus–insect cell protein expression: from dark horse to mainstream technology. J Gen Virol 96: 6–23
Yao T, Asayama Y (2017) Animal-cell culture media: History, characteristics, and current issues. Reprod Med Biol 16: 99–117
Zhang J, Kalogerakis N, Behie LA (1994a) Optimization of the physiochemical parameters for the culture of Bombyx mori insect cells used in recombinant protein production. Journal of Biotechnology 33(3): 249–258
Zhang Y, Zhou Y, Yu J (1994b) Effects of peptone on hybridoma growth and monoclonal antibody formation. Cytotechnology 16: 147–150

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Development of an Animal-Derived Component-Free Medium for Spodoptera frugiperda (Sf9) Cells Using Response Surface Methodology

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Abstract

Figures

Introduction

Materials And Methods

Cell line and cell culture

Media and supplements

Experimental design

Cell culture

Statistical analysis

Verification study

Study of the optimum cell density in the static culture

The combined effect of microfiltration and ultrafiltration of YE and ST on Sf9 cell growth

Adaptation of Sf9 cells to grow in suspension

Results And Discussion

Regression analysis of the selected model

Verification study

Optimum cell density in the static culture

Effect of microfiltration and ultrafiltration components of YE and ST

Adaptation of Sf9 cells to grow in suspension

Conclusions

Declarations

References

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