Optimization of Sf9 insect cell culture using deep-well blocks and C. necator-derived media supplements

To enable high-throughput optimization of insect cell culture conditions for protein expression, we adapted a miniaturized Sf9 culture system using 24-deep-well blocks (Bahia et al. 2005) and validated its performance against conventional 125 ml Erlenmeyer flask cultures (3 ml vs. 15 ml culture volumes respectively) with Sf-900 III medium supplemented with 5% FBS and 0.5% Penicillin/Streptomycin. Growth kinetics were comparable between the two formats, confirming that the scaled-down system maintains equivalent cell proliferation and viability (Fig. 1A). This miniaturized platform significantly reduces reagent usage, labor, and cell input while enabling the parallel testing of multiple conditions, making it well-suited for systematic optimization of baculovirus-mediated protein expression workflows.

Using this format, we initially evaluated the efficacy of C. necator-derived protein hydrolysates (CPH) and biomass extracts as alternative media supplements by performing a comparative growth analysis with established supplements, including algal extract (ISOGRO, Merck), YE, Spirulina extract (Silantes), and complete media (Sf-900 III, ESF921). For this analysis, ESF921 Δaa medium, a basal medium devoid of amino acids, was supplemented with 1% of either algal, bacterial or YE and further enriched with tryptophan, NH4Cl, glucose, & YE, following prior studies on the acid hydrolysis-based preparation of algal extract for improved growth outcomes (Sitarska et al. 2015). This analysis revealed robust proliferation occurred in complete media, while cells grown in C. necator-supplemented media achieved growth levels comparable to those in algal or YE enriched formulations.

However, cell proliferation in these 1% supplemented media (Fig. 1D-I) remained suboptimal compared to that in complete media (Fig. 1B, C). This reduced performance may stem from a lack of cellular adaptation to the unique composition of the bacterial extracts, which appeared to exert cytostatic rather than cytotoxic effects. Notably, 1% C. necator extract supplementation consistently supported cell viability and moderate growth without adverse effects, aligning with effective concentrations used in isotope-labeling protocols involving algal or yeast hydrolysates. These findings underscore the value of the deep-well block platform for scalable, resource-efficient optimization of insect cell culture conditions and highlight C. necator extracts as a promising, non-toxic supplement and may further enhance their utility for high-yield recombinant protein production and stable isotope labeling.

Fig. 1The alternative text for this image may have been generated using AI.

Sf9 cell growth and proliferation in various growth media. (A) Sf9 cell growth performance in 125 ml Erlenmeyer flask cultures (15 ml volume) versus 24-deep-well blocks (3 ml culture volume) in Sf-900 III medium supplemented with 5% FBS and 0.5% Pen/Strep. (B to I) Microscopic images of Sf9 insect cells cultured in media with different supplement conditions in a deep-well plate. The image shows cells with typical rounded morphology and uniform distribution, indicative of healthy proliferation. Cell density and morphology were assessed to determine the optimal supplementation strategy for isotopic labeling experiments in biomolecular NMR applications. Image captured at 100x magnification

Model protein (EPHA2) expression in various media

To evaluate the suitability of Cupriavidus-derived supplements for insect cell culture and their potential future use in isotope labeling workflows, expression of the model protein EPHA2 was analyzed in Spodoptera frugiperda (Sf9) cells using various media conditions (Fig. 2). EPHA2 carried a N-terminal Flag-His-Tev fusion tag and expression was assessed by Western blotting using an anti-His antibody.

Initial small-scale expression experiments were performed using in-house Sf9 cells (Invitrogen) in a 24-well format (3 ml culture volume). Cells were transferred into amino acid-depleted ESF921 (ESF921 Δaa) medium supplemented with different biomass- or extract-based supplements, including Spirulina biomass, Spirulina protein hydrolysate (SPH), Cupriavidus biomass, Cupriavidus protein hydrolysate (CPH), yeast extract (YE), algal extract, or complete commercial media (Sf900III or ESF921). For expression of EPHA2 protein, cells were infected with EPHA2 P2 recombinant virus. After three days of infection, equal amounts of total protein were loaded for each condition, and EPHA2 expression was detected by immunoblotting.

As shown in Fig. 2 (top left), EPHA2 expression was detectable across all supplementation conditions, indicating that each supplement provided at least partial nutritional support for recombinant protein expression. However, EPHA2 levels observed with Cupriavidus-based supplements (both biomass and CPH) were consistently weaker compared to fully supplemented media (Sf900III or ESF921). Among the various supplements, CPH supported detectable EPHA2 expression, with signal intensities comparable to SPH or algal extract, but lower than Spirulina biomass, YE and markedly lower than complete media. These data demonstrate that Cupriavidus-derived supplements are bioavailable to Sf9 cells, although initial expression levels were reduced under these conditions.

Because the in-house Sf9 cells were routinely maintained in nutrient-rich Sf900III medium prior to transfer into ESF921 Δaa medium, limited adaptation to nutrient-restricted conditions may have contributed to reduced expression levels. To address this, expression experiments were repeated using Sf9 cells obtained from Oxford Expression Technologies (OET), which are pre-adapted to ESF921 medium.

When OET Sf9 cells were used, EPHA2 expression levels increased across several supplementation conditions (Fig. 2, top right). Notably, expression supported by CPH was substantially improved compared to Cupriavidus biomass, indicating that the hydrolyzed form is more readily utilized by the cells. Under these conditions, EPHA2 expression in CPH-supplemented cultures was comparable to that observed with Spirulina biomass or SPH, although still lower than that achieved with complete commercial media.

To further examine whether additional factors could enhance protein expression, OET Sf9 cells were cultured with the same supplements in the absence (−) or presence (+) of fetal bovine serum (FBS). As shown in Fig. 2 (bottom left), inclusion of FBS led to a general increase in EPHA2 expression across most supplement conditions, including those containing CPH. Expression in complete media (Sf900III and ESF921) also remained robust under these conditions (Fig. 2, bottom right).

Together, these results indicate that Cupriavidus protein hydrolysate is taken up by Sf9 cells and can support recombinant protein expression, with performance improving significantly when cells are adapted to the basal medium and when additional supplements such as FBS are included.

Fig. 2The alternative text for this image may have been generated using AI.

Western blot analysis of EPHA2 expression in Sf9 insect cells using anti-His antibody. Expression of His-tagged EPHA2 was evaluated under various supplementation conditions in Sf9 cells. All samples were prepared identically from 3 ml of expression culture by resuspending a 500 µl cell pellet in 200 µl lysis buffer with protease inhibitor and cell-lysis with ultra sonification. Loaded 100 µg (Top left, Invitrogen Sf9 cell lysate) or 50 µg (Top left & Bottom) total protein from each lysate on SDS-PAGE. Western blot detection was performed using an anti-His antibody to assess recombinant protein expression levels. (Top Left): EPHA2 expression in Sf9 cells (Invitrogen) cultured with different media supplements, including Spirulina biomass, SPH, Cupriavidus biomass, CPH, YE, algal extract, Sf900III, and ESF921. (Top Right): EPHA2 expression in Sf9 cells (OET) under the same supplementation conditions as in the left panel. (Bottom Left): Comparison of EPHA2 expression in OET Sf9 cells in the absence (−) and presence (+) of fetal bovine serum (FBS) for each supplement condition. (Bottom Right): EPHA2 expression in complete media

Isotope enrichment of EPHA2 for NMR and Mass spectrometry labeling efficiency analysis

Isotope enrichment of EPHA2 for NMR and mass spectrometry analysis was achieved using stable isotope labeling with 15N and/or 13C. Following the optimization of EPHA2 expression in pre-adapted OET Sf9 cells, these cells were employed for isotope labeling experiments. Initial work utilized regular fetal bovine serum (FBS) with ESF921 Δaa medium due to its benefits in enhancing protein yield and cell growth.

For 15N labeling, OET Sf9 cells were initially grown to high densities in fully supplemented ESF921 medium, to be used for expression. After centrifugation of the desired number (~ 6 × 106 per ml) of OET Sf9 cells, the cells were transferred to ESF921 Δaa medium and were allowed to adapt for (two hours) nutrient-starved conditions. In the next step, the starved cells were transferred to ESF921 Δaa amino acid-free medium which was supplemented with 5% FBS, 0.5% penicillin/streptomycin (Pen/Strep), 1% unlabeled glucose, 1% 15N-labeled CPH, 20 mg/L 15N2-tryptophan, and 250 mg/L 15NH4Cl. Followed by infection with 2% EPHA2 P2 recombinant baculovirus. Cells were harvested after three days of post-infection. Uniformly 15N-labeled EPHA2 was purified from the cells with different purification steps(Gande et al. 2016) and used for NMR. The folding of the protein was assessed using 1H-1D and 1H, 15N HSQC 2D NMR spectra. High signal dispersion and uniform peak intensities confirmed that the EPHA2 protein was well-folded and homogeneous (Fig. 3).

Fig. 3The alternative text for this image may have been generated using AI.

1H-15N HSQC spectra of EPHA2. Two-dimensional 1-15N HSQC NMR spectrum of 15N-labeled EPHA2 kinase domain expressed and purified from a 250-ml culture grown in ESF921 Δaa medium supplemented with 15N Cupriavidus protein hydrolysate (condition III; Table 2). Spectra were acquired at 298 K on a Bruker 800 MHz spectrometer equipped with a cryo TCI 1H [13C, 15N] probe and measured in a 3-mm NMR tube. A 200 µl protein sample containing 130 µM EPHA2 kinase domain was prepared in 20 mM Tris-HCl (pH 8.0), 200 mM NaCl, 5 mM MgCl2, and 3 mM TCEP, with 10% (v/v) D2O included for field-frequency locking. Spectra were recorded with 40 scans per increment, 2048 complex points in the direct dimension (F2), and 224 increments in the indirect dimension (F1). Chemical shifts were referenced to 1 mM 3-(trimethylsilyl)-2,2,3,3-tetradeuteropropionic acid (TSP-d4) used as an internal standard

For 13C and combined 13C/ 15N labeling of EPHA2, ESF921 Δaa medium was supplemented with 5% FBS, 0.5% Pen/Strep, 1% 13C-glucose, 1% 13C-labeled or 13C/ 15N-labeled CPH, 20 mg/L 15N2-tryptophan, and 250 mg/L NH4Cl (unlabeled for 13C labeling; 15NH4Cl for 13C/ 15N labeling). The labeling protocol mirrored that of 15N incorporation, with cells adapted to amino acid-depleted conditions before being transferred to the isotope-enriched medium. After three days of infection with 2% EPHA2 P2 recombinant virus, the cells were harvested, and the isotopically labeled respective proteins were purified for further analysis by LC-MS measurements.

Determination of isotope enrichment by mass spectrometry (LC-MS)

The use of liquid chromatography-tandem mass spectrometry (LC-MS) for determining 15N and 13C isotope enrichment in proteins derived from insect or mammalian cells is crucial for metabolic studies and quantitative proteomics. This method allows precise detection of isotopically labeled amino acids incorporated into proteins such as actin, tubulin, or GAPDH, enabling insights into protein turnover and synthesis rates. The workflow typically involves culturing cells in media enriched with 15N - or 13C-labeled amino acids, followed by protein extraction, enzymatic digestion (e.g., trypsin digestion), and LC-MS analysis to quantify the isotope incorporation by measuring mass shifts in peptide fragments. This approach is widely used in stable isotope labeling by amino acids in cell culture (SILAC) and metabolic flux analysis. While mammalian cell cultures, such as HEK293 or CHO cells, allow controlled isotope labeling, insect cell systems like Sf9 or Sf21 are also effective for recombinant protein expression with isotope enrichment. This methodology provides high sensitivity and specificity, enabling differentiation between naturally occurring isotopes and experimental enrichment levels.

In this study, unlabeled (12C/14N) EPHA2 protein samples were first analyzed to establish a baseline peptide profile. Peptide fragments generated from tryptic digestion were subjected to LC-MS (Fig. 4A), and the resulting spectra were matched against a peptide database (Perez-Riverol et al. 2025). A total of 33 peptides were identified, yielding 30 unique peptide spectra and providing 79% sequence coverage of the EPHA2 protein, as indicated by bold regions in the sequence diagram (Fig. 4A). Retention times and m/z (mass-to-charge) envelopes were defined for each identified peptide, forming the foundation for subsequent isotopic incorporation analysis.

Using this reference dataset, the uptake of 13C and 15N was calculated by aligning spectra from labeled samples to the unlabeled controls based on retention time and m/z envelopes. For example, the peptide KEVPVAIK (C₄₁H₇₄N₁₀O₁₁; [M + 2 H]2+ = 442.515 m/z) was identified in the unlabeled sample with a retention time of 17.9 min (Fig. 4B). This information enabled detection of labeled isotopologues in subsequent runs, including 13C-labeled, 15N-labeled, and dual-labeled (13C + 15N) forms. Accurate frame alignment allowed the extraction of isotopic signals from these labeled peptides (Fig. 4C).

Fig. 4The alternative text for this image may have been generated using AI.

LC-MS-based determination of 15N/13C incorporation. (A) Sequence coverage of the target construct in a control, unlabeled LC-MS run used for peak assignment (detected segments in bold; sequence coverage of 79%, using 30 unique peptides detected with 514 PSMs; linker and TEV cleavage site not shown) (B) Total ion chromatogram and extracted ion chromatograms of selected peptides of a representative tryptic digest of EPHA2 used for isotopic envelope calculation. (C) Isotopic envelopes of selected peptides in unlabeled, 15N-, 13C- and 15N+13C-labeled samples. (D) Detected incorporation rates for indicated peptides (SF4Δaa ΔYE, replicate 1; 19 curated peptides, peptide values in grey, average in black). (E) Summed incorporation rates for EPHA2 for all quantified peptides per sample (dark grey bars: ESF921; light grey bars: SF4Δaa ΔYE/ SF4Δaa ΔYE ΔGSM)

Several medium compositions were tested to assess the efficiency of isotope enrichment using different nitrogen and carbon sources (Table 1). Isotope incorporation into recombinant EPHA2 was quantified by LC-MS, while protein yields were determined independently after purification using UV absorbance (NanoDrop). All yields were normalized to milligrams of purified protein per 100 ml culture to allow direct comparison across conditions (Table 2). Figure 4D and E presents the isotope incorporation levels of 15N, 13C, and combined 15N-13C enrichment in the EPHA2 protein (D596-G900) in OET Sf9 cells, determined through LC-MS.

In ESF921 Δaa medium supplemented with 15N YE and 0.027% 15NH4Cl, 15N incorporation reached 71.2% with a normalized protein yield of 3.38 mg/100 ml (Table 2, Sample I(c)). This condition provided a balanced reference, combining moderate isotope incorporation with relatively high protein yield.

Removal of YE from the basal medium (SF4 Δaa ΔYE), while supplying 15N YE and 0.027% 15NH4Cl, as complex nitrogen source, increased 15N incorporation to 78.5% (Table 2, Sample V(c)), but resulted in a pronounced reduction in protein yield (0.74 mg/100 ml), indicating that complete YE removal negatively impacts expression efficiency despite improved labeling.

To compare alternative complex nitrogen sources, SPH and CPH were evaluated under comparable conditions in SF4 Δaa ΔYE medium. Supplementation with 15N SPH resulted in 75.2% 15N incorporation (Table 2, Sample VI(c)), but protein yield remained low (0.74 mg/100 ml), similar to the ΔYE condition. This indicates that SPH supports efficient isotope incorporation but provides limited support for high-level protein expression.

In contrast, substitution of SPH with 15N CPH under the same basal conditions resulted in 75.7% 15N incorporation (Table 2, Sample VII) while increasing protein yield to 2.48 mg/100 ml. Thus, although SPH and CPH achieved comparable isotope incorporation, CPH supported substantially higher protein yields, indicating improved metabolic support for sustained protein synthesis.

The highest 15N incorporation (79.0%) was observed when 15N CPH was combined with 15NH₄Cl, 15N tryptophan, and 15N YE in SF4 Δaa ΔYE medium (Table 2, Sample VIII). Importantly, this condition also maintained a moderate protein yield, demonstrating that CPH can support both high isotope incorporation and acceptable expression levels when background unlabeled nitrogen is minimized but essential nutrients are retained.

When 15N CPH was used in ESF921 Δaa medium (Table 2, Sample III), 15N incorporation decreased to 63.1%, while protein yield remained moderate (~ 1.9 mg/100 ml). This reduction in enrichment reflects isotopic dilution from background nitrogen sources in the richer basal medium rather than inefficient utilization of CPH.

Table 2 Isotope incorporation levels of 15N, 13C, and combined 15N-13C enrichment in the EPHA2 protein in various growth media

For carbon labeling, use of 13C CPH in ESF921 Δaa medium (Table 2, Sample II) resulted in lower 13C incorporation and moderate protein yield, consistent with dilution by unlabeled carbon sources present in the basal medium.

In contrast, SF4 Δaa ΔYE ΔGSM medium supplemented with 13C-labeled CPH and 13C glucose (1%) achieved 69.0% 13C incorporation (Table 2, Sample IX). However, this condition resulted in a markedly reduced protein yield (0.34 mg/100 ml), indicating that extensive removal of background carbon sources strongly limits protein expression despite reasonable isotope incorporation. These results show that while carbon derived from CPH is readily incorporated into recombinant protein, carbon restriction imposes a stronger penalty on yield than nitrogen restriction.

Simultaneous 13C-15N labeling further accentuated the trade-off between isotope incorporation and protein yield. In ESF921 Δaa medium supplemented with 13C-15N CPH (Table 2, Sample IV), combined isotope incorporation remained low, with moderate protein yield (0.25 mg/100 ml), reflecting substantial dilution from unlabeled nutrients.

In SF4 Δaa ΔYE ΔGSM medium without starvation, combined 13C-15N labeling reached 70.6% incorporation (Table 2, Sample X), but protein yield was already reduced compared to single-isotope conditions. Introduction of a 1-hour starvation step prior to isotope supplementation further increased incorporation to 75.1% (Table 2, Sample XI) but resulted in an extreme reduction in protein yield (0.04 mg/100 ml).

In summary, CPH supported substantial isotope incorporation across 15N, 13C, and combined 13C-15N labeling conditions. For 15N labeling, CPH achieved isotope incorporation comparable to SPH and YE, while consistently supporting higher protein yields under matched conditions. Carbon and dual-isotope labeling using CPH were feasible but were associated with pronounced reductions in protein yield, particularly under stringent carbon restriction or starvation protocols. Overall, protein yield was more strongly influenced by basal medium composition and nutrient restriction than by the choice of hydrolysate, highlighting clear incorporation–yield trade-offs across labeling strategies.