Application And Evaluation Of Sericin Protein As A Serum Substitute Or An Additive In Cell Culture And Cryopreservation

In recent years,with the development of cell culture technology,the shortcomings of fetal bovine serum(FBS)become increasingly apparent.It is not only expensive but also poses biosafety problems due to viral infection.Therefore,the research and development of serum substitutes have attracted more and more attention.In the silk processing industry,sericin is usually used as a waste,but it has anti-UV,antioxidant and moisturizing activity.Because of its good biological compatibility,sericin is often used as skin and hair cosmetic and medical biomaterials such as 3D scaffold and surface modification materials.In this paper,we used silkworm cocoons(Bombyx mori)as the experimental material,sericin was derived from the cocoons and then hydroyzed by enzyme and alkaline.Firstly,the effects of enzyme hydrolyzed sericin on the growth of mouse fibroblasts(L929),four types of tumor cells(A549,7402,HepG-2,MCF-7)and hybridoma cells(3C2)were investigated.Secondly,the effect of alkaline hydrolyzed sericin on the cell activity,cell cycle and the collagen secretion of L929 were also stuied and analyzed in this paper.Simultaneously,the effects of alkaline hydrolyzed sericin on the growth,proliferation,cell cycle distribution and related gene expression of Chinese hamster ovary(CHO)cells and Human cervical cancer cells(Hela)were evaluated systematically.Finally,the effects of sericin on cryopreservation of L929 cells and rat pancreatic islets were also evaluated.The research of this paper mainly involved the following four aspects:(1)Cell cultures often require the addition of animal serum and other supplements.In this study,silk sericin,a bioactive protein,recovered from the waste of silk floss production was hydrolyzed into three pepsin-degraded sericin peptides with different ranges of molecular mass.Normal animal cells(L929),tumour cells(A549,7402,HepG-2,MCF-7)and hybridoma cells(3C2)were cultured systematically in FBS culture media containing sericin as a supplement or serum substitute for short term(1-4d).The culture test and microscopic observation of L929 cells showed that the smaller molecular weight of the degraded sericin is most suitable for cell culture.The cell culture results showed that with the degradation of sericin,for normal mouse fibroblast L929 cells,addition of 0.75 % sericin into FBS culture medium yields cell viability that is superior to FBS culture medium alone.When all serum was replaced by sericin,cell viability in the sericin medium could reach about one half of that in FBS medium.When in a medium containing a mixture of FBS: Sericin(6:4,v/v),the cell culture effect is about 80%.For the cultures of four tumour and one hybridoma cells,regardless of the molecular weight range,these degraded sericin peptides could substitute all serum in FBS media.The cell viability and proliferation of these tumour and hybridoma cells are equivalent or superior to that in FBS medium.In other words,cell viability and proliferation of these tumour and hybridoma cells in sericin media are more preferable to serum media.Therefore,the sericin membrane in the silk processing waste membrane recovery in the animal cell culture has a potential development value and application research.(2)The effects of enzyme hydrolyzed sericin on cell viability in the long term(1-16d)and alkaline hydrolyzed sericin on the viability,cycle and collagen of L929 cells were all researched by using alternative serum culture.The results showed that the activity of L929 cells was poor when FBS was replaced by the enzyme hydrolyzed sericin in the long term,it could not achieve the effect of FBS,which was consistent with the results of the previous study.But in the partial replacement,even at ninth day the cell viability value of this group(FBS: Sericin(1 mg/mL)= 1:9,v/v)could reached 75.4% of FBS.And the alkaline hydrolyzd sericin(1 mg/mL)is more suitable for the growth of L929 cells,even if the effect of all the alternatives is not ideal,but the cell viability of this group(FBS: Sericin(1 mg/mL)= 1:9,v/v)still achieved 89.7% of FBS.The following cell cycle and collagen content determination were all selected the 1mg/ml alkaline hydrolyzed sericin.The cell cycle was detected by flow cytometry.The percentages of G0/G1,S,G2/M cells were similar to that of FBS,indicating that there were no significant change and cytogenetic distortion in cell cycle after partial replacement of FBS.The expression of type I and type III collagen were detected by enzyme-linked immunosorbent assay(ELISA).The results showed that the expression level of type I collagen was also increased,while the ratio between type I and type III was 1.2 times that of FBS.We speculate that alkaline hydrolyzed sericin could be partially replaced with FBS to culture fibroblasts for medical cosmetic and other industries.(3)Alkaline hydrolyzed sericin was systematically evaluated in CHO cells and Hela cells as serum alternatives.Morphology,physiology and related genen expression of the two types of cells were studied.As a result,it was found that the cells cultured in the alkaline hydrolyzed sericin group showed similar cell morphology,similar or higher cell overall survival as compared with the cells cultured in FBS.Especially 15 ?g/mL alkaline hydrolyzed sericin on the fifth day,both cells showed a more intensive status.The absorbance of CHO cells reached 0.85 higher than that of FBS,whereas the absorbance of Hela cells was 1.44 higher than FBS(1.24).As well as a higher percentage of S phase and similar G2/G1 ratios,indicating a comparable or better cell growth and proliferation status in alkaline hydrolyzed sericin medium.Especially on the fifth day,the proportion of S phase in CHO cells was 28.9% nearly two times of FBS(12.9%).The proportion of S phase in Hela cells was 28.0% higher than that in FBS(23.9%).RT-PCR results showed that the differential gene expression of two cells between the two media were mainly enriched in the “inhibition of DNA binding(ID1,ID2)”,“cell cycle progression(FN1,MFSD4,MMP1)” and “cell proliferation and migration(INHBA,LCN2,CBY1,CXCL12,SCNN1A)”.The relative expression of CXCL12 gene in CHO cells increased to 3.1 times,while the relative expression of LCN2 gene in Hela cells increased to 2.75 times,indicating that these related genes are activated to promote cell growth and proliferation.In summary,both cells showed similar cell morphology,similar or higher overall cell viability in medium supplemented with 15 ?g/mL alkaline hydrolyzed sericin.High percentage of S phase(>28.0%)and similar G2/G1 ratio.Related genes such as(CXCL12,LCN2)were multiplied(3.1 times,2.75 times)activated,showing a considerable or better cell growth and proliferation status.The results of this study fully demonstrated that 15 ?g/mL alkaline hydrolyzed sericin can completely replace FBS in the culture of CHO cells and Hela cells.(4)The results of serum-free cryopreservation showed that the enzyme hydrolyzed sericin was beneficial to the cryopreservation of mouse fibroblasts(L929)and rat pancreatic islets,especially laboratory-made small molecule enzyme hydrolyzed sericin(10 mg/mL)could promote the secretion of collagen I in mouse fibroblasts.ELISA results showed that the expression of Collagen I of the laboratory-made small molecule sericin could reached to 21 ng/mL,but the FBS is 17 ng/mL.It is beneficial to the cryopreservation of rat pancreatic islet cells,the laboratory-made small molecule sericin could stimulates insulin secretion to a maximum of 34 ng/mL,and FBS group is 32 ng/mL.The results showed that the enzyme hydrolyzed sericin(10 mg/mL)could completely replace FBS for the the cryopreservation of mouse fibroblasts(L929)and rat pancreatic islets,and their specific mechanisms are still to be studied and discussed in the future.In summary,the effects of sericin on serum-free culture and cryopreservation of two different methods were studied in detail.The results of this study not only allowed us to have more understanding of sericin replacement of FBS for a variety of cell cultures,but also showed that small molecular weight sericin at low concentrations can partially replace of FBS or extra added in animal culture medium.This is a recycling and purification of sericin,which is often used as a waste in the industrial production of silk.The paper provided a potential new approach as a substrate for cell culture and medical biomaterials,which not only greatly reduces the contamination of silk processing,environment,but also can greatly reduce the cost of cell culture and cryopreservation and biosafety risk.It lay a good foundation for the sericin high value-added development and application,the sustainable development of the silk industry.