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A wide variety of highly viable seed cells are the source of the production of cell-cultured meat, however, after activating and differentiating into myoblasts, these cells lose their stemness and cannot grow permanently, and there are still many challenges to cell passaging, and continuous retrieval of cells from animal organisms is also contrary to animal welfare
.
Cell immortalization technology can enable cells to obtain the proliferation ability of continuous growth, long-term passage, and have the growth characteristics of unlimited proliferation, overcoming the situation
of aging and death after limited passage of general primary cells.
Immortalization of adult cells, including telomerase reverse transcriptase transfection, gene knockout restorative induction, four-small gemcqueline oxidation, and aflatoxin stimulation
.
However, most of the existing immortalized cell lines are mouse or human cells, which cannot be directly applied to cell culture meat systems, let alone food science research
.
Wang Shouwei (first author: Yang Feng), chief scientist of China Meat and Food Comprehensive Research Center, published a special column (cell culture meat column) article "Cell Cultured Meat Seed Cell Immortalization Induction Research" in the Journal of Chinese Food Science, Volume 22, Issue 12, which provides more efficient technical means for cell cultivation meat seed cell preparation by constructing a stable porcine myoblast cell immortalization induction vector, and lays a foundation
for the industrial production of cell culture meat seed cell acquisition.
Test method
Based on porcine myoblasts, the induction vector was constructed by telomerase reverse transcriptase, and after nuclear transfection into cells, cell induction verification
was carried out by immunofluorescent labeling, quantitative PCR, chromosome karyotyping and other methods.
Test results
Telomerase reverse transcriptase overexpression vector construction
Immortalization-induced overexpression vector maps
The detoxified lentivirus was used to construct the overexpression vector, and the constructed vector was verified by multi-primer PCR and sequencing, and the results were good, and no mutations occurred
in the sequence.
Puromycin screening, positive cell drug screening and cell viability testing
Apoptosis and proliferation curvesOn the basis of determining that the transduction titer titer is the highest efficiency of transducing cells, puromycin was used to screen positive cells to remove false-positive cells
.
Continuous passage verification found that the cells of the induction group had broken through the physiological limit and exceeded the "Hay flick" boundary (the maximum number of cell divisions), proving that the induction of cell immortalization in this study had achieved initial success
.
Flow cytometry purification of positive cells and identification of cell positivity rate
Flow cytometry identifies the degree of purification of positive cells
Flow cytometry cell identification was carried out on the 2nd, 4th, and 6th days of induction cells to add puromycin, and the screening efficiency of positive cells was detected, and it was found that with the extension of time, the proportion of positive cells continued to increase, and the proportion of positive cells on the 6th day was 100% (there were a very small number of negative cells outside the circle, the proportion was close to 0), and the negative cells had been screened
out by puromycin.
Identification of genetic stability of immortalization-induced cell lines
Karyotyping
Karyotyping analysis of cells that passed through the 50 passages after induction showed that the central centromeres and secondary scars of chromosome 1 were deeply stained, chromosome 2 had 4 deep bands in the short arm and 6~7 deep bands in the long arm, divided into 3 regions; Centromeres on chromosome 3 are strongly stained, with 1 distinctly broad shallow band at the middle end of the short arm and one long arm; The other chromosomes did not produce significant abnormalities
in morphology, length, and arm ratio.
Telomerase reverse transcriptase and telomere tetramer expression identification
The expression of hnRNP A2 protein was positively correlated with telomerase activity in immortalization-induced cells, and the expression of hnRAP A2 promoted telomere lengthening
.
In addition, in adult cells, telomerase cannot be detected, while in this induction cell line, telomerase expression can be detected, proving that the induction of cells is effective
.
Telomerase reverse transcriptase and hnRAP protein expression activity identification
Compared with the length of telomeres, the speed at which telomeres become shorter is of greater significance.
The results showed that the relative telomerase length of immortalization-induced cells was significantly higher than that of the control group, which revealed that the efficiency of cell immortalization induction was high, and the cell passaging ability and cell viability were significantly higher than that of the control group
.
In addition, the expression of hnRAPC gene was significantly higher than that in the control group, revealing the activation function of the protein in telomeres, and the activity of inducing histacyte proteins was significantly higher than that in the control group, indicating that the induction of immortalized cells was successful and the cell passage ability was significantly improved
.
Discussion
As an emerging science and technology, although the research time of cell culture meat is relatively short, it has a deep research foundation
as a multidisciplinary cross-integration technology such as cell biology, tissue engineering and food science.
In the process of cell culture meat research and development, obtaining corresponding adult cells that can proliferate indefinitely is an important guarantee
to solve the source of cells in the process of industrialization.
At the gene level, the methods of cell immortality mainly include SV40 large T antigen method, telomerase reverse transcriptase method, HPV16 E6 method, proto-oncogene and tumor suppressor gene
.
As a food science research, the seed cells used in cell culture meat first need to exclude human factors, while ensuring the activity and functions of cells, and more importantly, the induction of cell immortality can not interfere with the normal use of various seed cells, that is, uncertain factors
can not be introduced.
of telomerase.
At the same time, it interacts with hnRNPA2 protein, opens the telomere G-quadruplex structure, exposes the telomere 3' end base, promotes its pairing with the telomerase RNA template, thereby enhancing the catalytic activity and progressivity of telomerase, and effectively maintaining the length of telomerase, which is significant in reducing (negative regulation) the rate of telomere shortening, so that telomeres maintain a certain length, thereby realizing cell immortization
.
In addition to telomerase reverse transcriptase, the immortalized cells obtained by this method do not introduce new influencing genes to ensure the normal expression level and function
of cells.
After 180 passages, the cells still have high proliferation and differentiation efficiency, and the cell stability is high, and there is no unpredictable mutation.
ConclusionIn this study, the immortalization induction vector of porcine myoblasts was constructed, and the cell immortalization induction scheme and research materials were explored.
Positive cell lines were obtained by nuclear transfection and positive cell flow cytometry screening; Verify the stability of cells by quantitative PCR, immunofluorescent labeling, karyotyping, etc.
; Positive cells were continuously subcultured to verify that cells could be passaged for more than 200 passages
.
The implementation of this study explores the theoretical basis for the development of cells of cultured meat seed cells, provides the cell materials for industrial production, and provides the leading conditions
for the industrial production of cultured meat.
Introduction by experts
Wang Shouwei is a professor-level senior engineer
Chief Scientist, China Meat and Food Comprehensive Research Center
Chief Scientist, Beijing Institute of Food Science
Wang Shouwei, professor-level senior engineer, chief scientist of China Meat and Food Comprehensive Research Center and Beijing Institute of Food Science, chairman of the Meat Processing Industry Technology Innovation Strategic Alliance, and standing director ofthe Chinese Society of Food Science and Technology.
He has been engaged in the research of meat science and technology and food safety for a long time, and is committed to the research of major key common technical issues in the meat industry and the transformation and industrialization of scientific and technological achievements, so as to promote the development of
biological cultivated meat research in China.
He has presided over and undertaken more than 20 projects/projects such as the National Key R&D Program, the National "863" Program, and the National Natural Science Foundation of China, published more than 190 scientific and technological papers, obtained 20 authorized invention patents, formulated and revised 9 international/national/industry standards, published 10 monographs, won 1 second prize of National Science and Technology Progress Award, 10 first prizes of provincial and ministerial level Science and Technology Progress Award (first completer)
The article comes from the WeChat public account of "China Journal of Food Science"
