Evaluation of chicken embryo extract and egg yolk extract as alternatives to basic cell culture medium supplement

Abstract

Background

Fetal calf serum (FCS), an existing cell culture supplement, is effective but has several drawbacks, including being expensive, requiring a lengthy process of production, and requiring a hard currency. With this in mind, we planned to evaluate chick embryo extract and egg yolk extracts in cell culture as alternatives to fetal calf serum (FCS).

Methods

Specific pathogen-free eggs were purchased from the National Veterinary Institute, Bishoftu, Ethiopia, and incubated in a humidified incubator at 37 °C for 11 days. Egg yolk extract (EYE) and chick embryo extract (CEE) were collected after the egg was opened with caution not to destroy the yolk sack or the chick embryo itself. Chick fibroblasts and Vero cells were cultured in minimum essential medium (MEM) supplemented with egg yolk extract or chick embryo extract at ratios of 0:10, 1:9, 2.5:7.5, and 5:5% fetal calf serum.

Results

Fibroblast cell attachment was better in media supplemented with 5% CEE and 5% FCS. The confluency was also greater than 50% at this concentration. Vero cells cultured with 5% CEE and 5% FCS also exhibited very good cell attachment and a confluency of up to 70%. Viability and confluency were also observed at 5%:5% ratios of 50 and 70%, respectively.

Conclusion

This investigation evaluated these two extracts as cell culture supplements and revealed promising results as alternatives to fetal calf serum. The limitation of this study is that it only used two cell types and additional cell lines, and different ratios should be tested. With the above findings, further research using different cell lines, ratios and conditions is warranted.

Introduction

Chick embryo extract (CEE) is derived from whole chick embryos and is known to contain essential proteins, growth factors, and hormones that are beneficial for cell culture [1]. CEE has been used to culture neural crest and neuroepithelial stem cells [1, 2]. In addition, CEE supplement has been shown as growth proliferation of human muscle stem cells [3]. Improvement of immune system performance was observed as a result of enhancement of lymphocyte proliferation supplemented by CEE [3]. Ongoing research is focused on understanding how a number of growth factors present in CEE synergistically contribute to enhancing cell growth in vitro [4]. The medium supplemented with the low and large molecular weight fraction of CEE promoted full differentiation and growth of cartilage and pigmented retina cells [5].

In chick embryos, a number of growth factors have been identified, including nerve growth factor (NGF), epidermal growth factor (EGF), and stem cell factor that are useful for cell proliferation (SCF) [6, 7]. These substances found in the CEE have been shown to enhance numerous physiological processes, including preserving neuronal survival and repairing tissue damage. Therefore, to generate a variety of stem cells, animal embryonic extract (chicken embryonic extract) is used as a growth factor cocktail [8].

Furthermore, it is believed that chicken embryo extract can provide functional and structural proteins to the medium used for in vitro cell development [9, 10]. The expansion and maturation of embryonic stem cells, neuronal cells, and neuroepithelial cells in culture have been promoted using these supplements [11, 12]. The addition of chick embryo extract (CEE) to cell culture medium provides essential growth factors for neural crest stem cells (NCSCs), which improve the shape and growth rate of cell lines [13, 14].

Researchers have explored alternative cell culture supplements to reduce or replace the use of fetal calf serum (FCS). One promising option is the use of egg and chick embryo extracts, which contain substances and factors necessary for animal cell growth and differentiation. Previous studies have demonstrated the stimulatory effects of these extracts on the development of animal cells in culture, including hybridoma cells and fibroblast cultures. These findings suggest that the liquid derived from various compartments of fertilized eggs can serve as a suitable supplement to replace FCS in cell culture media [15, 16].

Therefore, considering the advantages of egg yolk extract and chick embryo extract mentioned above and their cost-effectiveness, EYE and CEE are considered as a promising supplements for creating favourable conditions for cell growth. This would help to act as an alternative source of supplement due to its accessibility, easy to produce and utilizes non-intrusive method of collection. This research focused on evaluating the efficiency of egg yolk extract (EYE) and chick embryo extract (CEE) as alternatives to fetal calf serum supplements in basic cell culture media.

Materials and methods

Egg incubation, egg yolk, and chick embryo extraction

Specific pathogen-free (SPF) eggs were purchased from the National Veterinary Institute, Bishoftu, Ethiopia, and incubated in a humidified incubator at 37 °C for 11 days. Briefly, SPF eggs were cleaned with 70% ethanol for 30 s and gently and carefully dried with clean tissue paper towels. The eggs were opened carefully, and the egg yolks were harvested with a syringe. Egg yolk extract (EYE) was diluted 1:5 (v/v) with PBS, homogenized, and subsequently frozen for 72 h (-20 °C). The slowly thawed extract was centrifuged, and the clear supernatant was stored at -20 °C until use. The chick embryo extract (CEE) was collected after the incubated egg was opened with caution not to destroy the yolk sack or the chick embryo itself. The beginning of the umbilical cord was searched, and the clear wrapping was opened without destroying the yolk sack or any small vessels. Then, the umbilical cord was dissected with precise clean forceps. The embryo was removed from the yolk sack and placed in minimal essential medium at 4 °C. The plunger was removed from a 60 mL sterile syringe. Briefly, 10 embryos were transferred to a 60 mL syringe. Then, the chicken embryo was macerated by pushing down the plunger. The macerated mass was collected in prepared Corning tubes half-filled with DMEM at a ratio of 1:1 (macerated mass: DMEM). The mixture was placed on a centrifuge for 45 min at 4 °C. After centrifugation, the CEE was filtered, and three phases were observed: the upper phase consisted of a dingy liquid phase with fatty fragments, the clear liquid phase was observed in the middle, and the pellet was observed at the bottom of the centrifugation tube. The clear central liquid phases were collected and used as a cell culture supplement.

Sterility of the extracts

The sterility of the extracts was checked for any bacterial contamination by performing bacteriological tests. The bacteriological test performed was streaking in to a tryptose broth to check the presence or absence of bacterial growth. No growth of bacteria was found after 24 h of incubation. For the presence of virus in the egg, independent cell culture was performed, and the presence or absence of cytopathic effects was checked.

In vitro cell culture

Vero cells (a kind gift from the National Veterinary Institute, Ethiopia) and primary fibroblasts (prepared from our own primary cells) were used for this experiment. These cells were cultured with different percentages of egg yolk extract and chick embryo extract as alternative to fetal calf serum. As a positive control, we used 10% commercial calf serum-supplemented medium. Briefly, Vero cells were cultured in minimum essential medium (MEM) supplemented with egg yolk extract or chick embryo extract. The supplement was set at ratios of 0%:10%, 1%:9%, 2.5%:7.5%, and 5%:5% of the fetal calf serum supplement (Table 1). The cells were grown in 25 mm² tissue culture flasks (VMR North America lot no. 100062889) that were placed in a 5% CO₂ atmosphere in a humidified incubator at 37 °C. Cell proliferation was determined by visualization of cell growth, confluence, and attachment under an inverted microscope and cell counting before and after treatment. In addition, staining of the cells was performed using trypan blue. The plates that were cultured with egg yolk extract and chick embryo extract supplements were compared with the plates that were cultured with fetal calf serum supplements.

Table 1 Different percentages of yolk extracts or chick embryos used for the culture of Vero cells and primary cell (fibroblasts)

Chicken fibroblast culture

In addition to the Vero cell culture described above, chicken fibroblasts were prepared from SPF eggs incubated for 11 days as follows. The incubated eggshell above the air sac was broken aseptically with scissors or forceps, taking care not to contact the underlying membrane. Then, using sterile forceps, the membrane was removed, the embryo was exposed, and the embryo was slowly removed to remove it from the yolk and placed into a sterile petri dish. Using a fresh pair of sterile scissors, the head, wings, feet and body cavity contents were dissected from the embryo and placed into a 50 ml conical tube. At this stage, the contaminating blood/yolk was removed by the addition and removal of 10 ml of 1x PBS with calcium and magnesium. Following this maceration of the embryo using dissecting scissors, 50 ml of 1x PBS supplemented with calcium and magnesium was added to the tube. This tube was inverted to wash the embryo, and large pieces of the embryo were allowed to settle to the bottom of the tube. After the addition of PBS, 25 ml of thawed 0.25% trypsin (HI media lot TC215) and 75 ml of versene solution (Caroerba reagent lot 102548) prewarmed to 37 °C were added. The tube was gently shaken for 15 to 20 min at 37 °C. Using a pipette, the supernatant containing the cell suspension was transferred to a tube containing 25 ml of 1× complete MEM (Sigma Englnad Lot SLCH151559). The serum in the medium was supposed to inactivate the trypsin. Centrifugation at 500 × g for 10 min was performed at room temperature. The supernatant was removed, and the cell pellets were resuspended in 20 ml of 1× complete MEM (Sigma Englnad Lot SLCH151559). At this stage, the pellet was loose, so care must be taken when removing the supernatant. An aliquot of the cell suspension was diluted 1:10 in PBS containing calcium and magnesium. The number of cells was counted using a hemocytometer [17]. This procedure was used to collect or confirm the presence of ∼10⁶ to 10⁷ cells/ml.

Culture of chick embryo fibroblasts

Flasks of 75 cm² were used to culture 1 × 10⁶ cells, and 30 ml of 1× complete MEM (VMR North America lot no. 100062889) was placed in a 5% CO₂ atmosphere in a humidified incubator at 37 °C for 24 h. The supplement was set at ratios of 0%:10%, 1%:9%, 2.5%:7.5%, and 5%:5% FCS supplement (Table 1). Cell proliferation was determined by visualizing cell growth, confluence, and attachment under an inverted microscope and performing cell counting before and after cell proliferation. In addition, staining of the cells was performed using 0.1 mL of trypan blue stock solution to 0.1 mL of cells cell immediately after trypsinzsation according to (https://bpsbioscience.com/trypan-blue-staining-protocol). Then, 25 ml of 0.25% trypsin (HI media lot TC215) and 75 ml of versene solution (Caroerba reagent lot 102548) were mixed together.

Cell attachment and confluency

The flasks were removed from the incubator, and the microscope was cleaned using 70% ethanol to avoid contamination. Cell attachment was evaluated under a stereomicroscope to determine cell adherence. On the other hand, confluency was checked to determine how much the culture vessel surface was covered with cells.

Cell counting

Trypsin was added to the monolayer after decanting with PBS, and the mixture was then incubated for 3 min to detach the cells from the flask. The trypsin was decanted and replaced with the appropriate amount of 1× complete MEM (Sigma Englnad Lot SLCH151559). The cells were then resuspended by pipetting up and down. Ten milliliters of the mixed solution was removed, and the solution was gently mixed on a cell-counting slide. The automatic cell counter device was adjusted, and a slide was inserted into it. A fine adjustment was made until the cells appeared to be white in color on the screen, after which counting started.

Cell staining

Ten millilitres of the cell suspension was transferred to a 96-well plate using a pipette, and 10 ml of trypan blue was added. This dye exclusion test is performed to identify the number of viable cells available in a cell suspension. The principle behind this test is that live cells have an intact cell membrane that excludes dyes such as eosin or trypan blue, whereas dead cells accept this kind of dye. In this test, the cell suspension is mixed with a dye, which is then examined under a cell counter. Therefore, viable cells appear to have a clear cytoplasm, whereas nonviable or dead cells appear to have a blue cytoplasm [17].

Results

Cell attachment and confluency of primary cells

In this study, we used four different concentrations of egg yolk extract (EYE) and chick embryo extract (CEE) as alternatives to supplement cell culture media. The concentrations used in these experiments were full replacement of fetal calf serum (100%) and 50% replacement (5%:5%) of fetal calf serum by egg yolk extract or chick embryo extract (CEE) and 90% chick embryo extract and 10% FCS. The results indicated that cell attachment was greater in media supplemented with 50% CEE and 50% FCS. The confluency was also greater than 50% at this concentration (Table 2). Cells that were cultured with chick embryo extract-supplemented media supplemented with 10% fetal calf serum also showed promising results, with comparable cell attachment and confluency to those of the control (Fig. 1).

Fig. 1

Primary cell culture was supplemented with different concentrations of egg yolk extract and chick embryo extracts. A: Control (10% FCS) (10x), B: 10% CEE (10x), C: 10% EYE (10x), D: 5% EYE: 5% FCS (10x), E: 5% CEE: 5% FCS (10x), F: 9% EYE: 1% FCS (10x)

Cell counting and viability for vero cell

The cells were counted before and after the different cultured cell types were seeded. This counting was performed to determine the number of viable and dead cells after replacement with different concentrations of fetal calf serum. In this case, viable cells were counted before culture to be used as a control and after culture at different concentrations. The number of viable cells counted before was 1.04.1 × 10⁶/ml and after 5%:5% replacement of fetal calf serum with CEE was 6.1 × 10⁴/mL, and the number of dead cells was 4.8 × 10⁴/mL (Table 2). The confluency and viability of the cells cultured with 5% fetal calf serum and 5% CEE was 62% and 55%, respectively. On the other hand the confluency and viability of primary cells cultured with 5% EYE and 5% fetal calf serum was 40% and 31%, respectively (Fig. 2).

Table 2 Primary cells (chick embryo fibroblasts) at different concentrations after washing with PBS

Fig. 2

Primary cells viable cell and dead cell counts (A), primary cells viability, and confluency (B) after the cells were washed with PBS and cultured with different concentrations of EYE and CEE

Cell attachment and confluency of vero cells

Similar to the above primary cell culture methods, we also cultured Vero cells (continuous cell lines) using different concentrations of egg yolk extract (EYE) and chick embryo extracts (CEE) as a supplement to the cell culture medium alternative to fetal calf serum. The chick embryo extract-supplemented medium used to culture Vero cells exhibited good cell attachment and confluency compared with Vero cells cultured with chick embryo extract-supplemented cells. The foetal calf serum was fully replaced with chick embryo extract (Fig. 3B) as a supplement to the cell culture medium. Compared with those of the control, cell attachment and confluency were found to be promising (Fig. 3A). The Vero cells cultured with 5% CEE and 5% FCS also exhibited very good cell attachment and a confluency of up to 70% (Table 3). The other concentration was 1% FCS/9% CEE, and as shown in Fig. 3F, the cells exhibited poor attachment and confluency.

Fig. 3

Vero cells cultured with different concentrations of yolk and embryo extracts. A: Control (10% FCS) (10x), B: 10% CEE (10x), C: 10% EYE (10x), D: 5% EYE: 5% FCS (10x), E: 5% CEE: 5% FCS (10x), F: 9% EYE: 1% FCS (10x)

Cell counting and viability for vero cell

The cells were counted before and after the different cultured cell types were seeded. This counting was performed to determine the number of viable and dead cells after replacement with different concentrations of fetal calf serum. In this case, viable cells were counted before culture to be used as a control and after culture at different concentrations. The number of viable cells counted before culture was 9.15 × 10⁵/ml and after 5%:5% replacement of fetal calf serum with CEE was 8.1 × 10⁵/mL, and the number of dead cells was 1.9 × 10⁵/mL (Table 3). The confluency and viability of the cells cultured with 5% fetal calf serum replacement and 5% CEE was 51%, respectively. On the other hand vero cells cultured with 5% EYE and 5% fetal calf serum was 50% and 25%, respectively (Fig. 4).

Table 3 Vero cells at different concentrations after washing with PBS

The viability and confluency of cells supplemented with different concentrations of EYE and CEE ranged from 51 to 16%. CEE-supplemented media at a 5%:5% ratio resulted in 51% viability and 70% confluency, which is close to that of the 10% FCS-supplemented Vero cells. In this table the data under attachment: Better means very few dead cells flowing in the medium and moderate means there are cells attached but few dead cells are flowing in the medium (Fig. 4).

Fig. 4

Vero cells Viable cell and dead cell counts (A), Vero cells viability, and confluency (B) after the cells were washed with PBS and cultured with different concentrations of EYE and CEE

Discussion

Chick embryo extract and egg yolk extract have been used by different researchers as alternative sources of supplements to cell culture media for in vitro cell growth. In our study, we used criteria such as cell viability, confluency and attachment to analyse the effects of both extracts on primary cells and continuous cell lines (vero cells). In this study, on Vero cells supplemented with 5% CEE, 70% confluency with good attachment was observed. The cell attachment was good, with 51% cell viability compared to that of the control. This finding is in agreement with the finding of Christman et al., that used CEE for cell growth [18], who used CEE treatment for the slow-growing SC-1 cell line, which showed a good improvement in growth rate. In our study, 10% EYE (not shown in the figure) supplemented media used for primary and Vero cell culture resulted in 5% confluency and poor cell attachment, and no cell growth was observed. Our findings disagree with those of Fuji and Gospodarowicz, who reported that the final cell densities of cultures grown in egg yolk extract-supplemented medium and serum-supplemented medium were the same. The discrepancy between our findings might be attributed to the type of cell used. At other supplement ratios, our morphological findings were comparable to the findings that reported similar morphologies with different supplements used [19].

At 50% confluency, 5% FCS:5% EYE was used, and 25% cell viability was reached. When 1% FCS:9% EYE was used, 20% confluency and 16% cell attachment viability were observed. This finding agrees with the finding of Joglekar et al., [20] that egg yolk extract in the presence of low serum concentrations promoted differentiation and maintained cell viability. The disagreement might arise from the use of different cell types and different purification and formulation procedures used for egg yolk extracts. In general, less supplementation (1:9) of egg yolk extract and chick embryo extract improved cell viability, confluency and attachment but increased the ratio of CEE- and EYE-supported cells with good viability, confluency and attachment.

Conclusions

Overall, the results of this research imply that extracts from chick embryos and egg yolks might be an alternative to FCS in cell culture. In our study, we found that chick embryo extract was superior to egg yolk extract at different ratios. Fibroblasts (primary cells prepared by an investigator) and Vero cells were used in this study. Cell attachment, cell viability and confluency were better at higher ratios of serum to chick embryo extract/egg yolk extracts. Further investigations are advised to determine their suitability for particular cell types to recommend scientifically sound FCS substitutes for cell culture investigations. The limitation of this study is that it only used two cell types and additional cell lines, and different ratios should be tested.