The Preparation Of Soybean Starch Microcarriers And Its Application In Cell Culture

The starch microspheres has been widely explored in the applications of slow-release drug,the embedding agent and adsorbent.However,the application of starch microspheres in adherent animal cell culture was rarely reported.The preparation of the microcarrier for cell culture using starch has the unique advantage.This thesis studied the preparation of suitable microcarrier with low cost starch as material,for the animal cells adherent culture.The purpose is to develop high valuable,biodegradable,and enzyme digestible to harvest cells,non-animal derived material starch microcarrier,avoiding the disadvantages to use animal derived material in biomedical field and the high cost of microcarrier.First,the research progress of microcarrier,the basic properties of polymer microspheres,the preparation method of starch microspheres and their application progress and present situation were introduced.Moreover,this thesis discussed the feasibility to apply the starch microspheres in the adherent animal cell culture.Based on the previous research of preparation for starch microspheres,the preparation technology and parameter optimization were studies to optimize the process conditions of preparation of starch microspheres.The results showed in the conditions of the machine oil and isooctane as oil phase,the 6%concentration of gelatinized and aqueous alkali starch solution as water phase and polyethylene glycol diglycidyl ether as the crosslinking agent,the optimal parameters for the preparation were as follows:the oil/water volume proportion of 1:4,the mixing speed of 500 RPM,and the crosslinking agent dosage of 10%.The preparation of crosslinked starch microspheres can tolerate high temperature of 121? for 30 min in the autoclave.By using orthogonal test and the ligands selection,the surface of starch microspheres was coupled with diethyl amino ethyl(DEAE),and the surface of starch microspheres and commercial Cytodex 1 microcarrier have the approximation of charge.The basic physical and chemical characteristics of crossing-linked starch microspheres were analyzed.The starch microcarrier was used to cultivate the BHK-21,Marc-145 and MDCK,which are common adherent animal cells used in three kinds of vaccine production,meanwhile,these tree cells were cultivated using Cytodex 1 microcarrier as control group.The results showed that the preparation of starch microspheres after coupling DEAE,are able to tolerate high temperature.in the autoclave.These three species of the cells have a good performance of attachment and proliferation when they grew on the starch microspheres,which were coupled the DEAE,and have the similar particle size as Cytodex 1.Under the same condition of suspension culture,the quantity of cells growth on starch microcarrier were comparable to that on Cytodex 1 microcarrier,these three species of cell proliferation rate achieves maximum in the culture for 144 h.Among them,the maximum proliferation of the BHK-21 cells on the starch microcarrier was 2.5×106 cells/mL,slightly higher than Cytodex 1(2.2×106 cells/mL).The maximum proliferation of Marc-145 cells on the starch microcarrier was 1.4×cells/mL,and that of Cytodex 1 was 1.6×106 cells/mL,the latter is slightly higher than the former.The maximum proliferation of MDCK cells on the starch microcarrier was 2.2×106 cells/mL,and that of Cytodex 1 was 2.4×106 cells/mL,which is slightly higher than the former.The cell growth curve and metabolic index curve on the microcarrier produced in this research are also similar to those on commercialized Cytodex 1 product.In conclusion,the preparation of starch microspheres by coupling DEAE,can be used as BHK-21,Marc-145 and MDCK adherent cells in the microcarrier suspension culture.The performance of cell culture were similar to Cytodex 1.Thus,the starch microcarrier have a good potential and application prospect.