CN117511851A - Application of hydrogel scaffold in preparation of cell culture meat - Google Patents

Application of hydrogel scaffold in preparation of cell culture meat Download PDF

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CN117511851A
CN117511851A CN202410006080.5A CN202410006080A CN117511851A CN 117511851 A CN117511851 A CN 117511851A CN 202410006080 A CN202410006080 A CN 202410006080A CN 117511851 A CN117511851 A CN 117511851A
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hydrogel
cells
polypeptide
culture
cell
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CN117511851B (en
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李石磊
梁俊
李莹莹
杨峰
刘屹森
梁小娟
唐铎
祁宇
陈嘉璇
董圣艳
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China Meat Research Centre
Tianjin University of Science and Technology
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China Meat Research Centre
Tianjin University of Science and Technology
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Abstract

The invention relates to the technical field of food production and processing and cell culture, in particular to application of a hydrogel bracket in preparation of cell culture meat. The hydrogel scaffold comprises polypeptide, thickener and water; the polypeptide is obtained by inserting an RGD sequence into an amino acid sequence shown in SEQ ID NO. 1. The polypeptide forms a nanofiber network structure in a hydrogel system, and is used as a bracket for cell culture, so that cells can proliferate in a large amount in the bracket. Meanwhile, with the adjustment of the concentration of the polypeptide in the hydrogel, the cells can be differentiated into tissue cell forms close to slaughtered meat, and the obtained cell culture meat has better taste and nutrition and high commercial value.

Description

Application of hydrogel scaffold in preparation of cell culture meat
Technical Field
The invention relates to the technical field of food production and processing and cell culture, in particular to application of a hydrogel bracket in preparation of cell culture meat.
Background
With the push of population and income increase, the total consumption of meat is in a continuously rising situation, and the traditional animal husbandry cannot meet the requirements of human beings on meat in the future, so that the problem of environmental pollution caused by the traditional animal husbandry is not quite a small one. The cell culture meat is also called as biological culture meat, cell culture meat, clean meat and the like, and is a novel meat food which is prepared by controlling the rapid proliferation, directional differentiation and collecting and processing of animal cells in an in-vitro culture mode. Numerous studies have shown that the production process of cell-cultivated meat will occupy less land area, consume almost negligible fresh water resources, have significant advantages in terms of greenhouse gas emissions, and at the same time, cell-cultivated meat can avoid pathogenic pollution and drug residue risks in animal feeding and slaughtering links, and is currently being developed towards rapid iteration of industrialization.
A qualified meat culture not only needs to harvest a large amount of cells in the early stage, but also needs to induce the cells to differentiate after harvesting the cells and finally form tissues, so that the product is as close as possible to the slaughter meat which we eat at present. Traditional cell culture schemes cannot meet the demand of meat cultivation on cell number.
The production process of cell cultured meat mainly comprises four steps according to the definition of the United states FDA (Food and Drug Administration, FDA for short): cell bank construction, cell proliferation, cell differentiation and cell harvesting. The industrial development of cell culture meats still presents some important technical hurdles that limit the scale-up and commercial progress of their production: (1) cell culture and differentiation technology studies; (2) development and optimization of non-animal derived media; (3) development of a high-efficiency bioreactor; and (4) is suitable for the research and development of the 3D growth bracket of the cells in vitro. Currently, research progress in cell culture of meat cell scaffolds mainly includes the following two types, one is to realize in vitro 3D culture and differentiation of seed cells through the development of novel scaffolds that promote cell adhesion growth. Team Shulamit Levenberg developed a textured soy protein scaffold for 3D culture of bovine skeletal muscle tissue with a maximum porosity of 56% but only 15% of the small voids with a diameter of less than 50 μm using soy protein. The other is to realize the in vitro three-dimensional culture of cells through the utilization and development of microcarriers, and Mark J.post team developed a technology for the in vitro large-scale amplification of bovine myoblasts based on the traditional commercial microcarriers Cytodex 1 and synthamax II, which greatly improves the culture density of the bovine myoblasts compared with the planar culture. However, although the scaffold material can realize proliferation or differentiation of different seed cells, several outstanding problems cannot be thoroughly solved: 1) a low porosity and cell fraction of the scaffold material, 2) a risk of food safety of the scaffold material, 3) a high production cost and difficult scale up of the scaffold material, 4) an animal-derived raw material source of the scaffold material. These factors greatly limit the development of industrial application value of the material.
In view of this, the present invention has been made.
Disclosure of Invention
In order to solve the technical problems, the invention provides the application of the hydrogel bracket in the preparation of cell culture meat, cells can proliferate in a large amount in the hydrogel bracket, and the edible meat obtained by culture has stable quality and higher commercial value in commercial production of the cultured meat.
Specifically, the technical scheme of the invention is as follows:
in a first aspect, the present invention provides the use of a hydrogel scaffold for the preparation of cell culture meat, animal cells being cultured using the hydrogel scaffold; the hydrogel scaffold comprises a polypeptide, a thickener and water; the polypeptide is obtained by inserting an RGD sequence into an amino acid sequence shown in SEQ ID NO. 1. SEQ ID NO.1: FFFFFGSIIPGGVVGPGGVG.
The polypeptide forms a nanofiber network structure in a hydrogel system, and is used as a bracket for cell culture, so that cells can proliferate in a large amount in the bracket. Meanwhile, along with the adjustment of the concentration of the polypeptide in the hydrogel, cells can be differentiated into tissue cell forms close to slaughter meat, and the obtained cell culture meat has balanced nutrition components and high engineering application value.
Preferably, the amino acid sequence of the polypeptide is selected from at least one of the following sequences (SEQ ID NO. 2-19):
SEQ ID NO.2：RGDFFFFFGSIIPGGVVGPGGVG，
SEQ ID NO.3：FRGDFFFFGSIIPGGVVGPGGVG，
SEQ ID NO.4：FFRGDFFFGSIIPGGVVGPGGVG，
SEQ ID NO.5：FFFRGDFFGSIIPGGVVGPGGVG，
SEQ ID NO.6：FFFFRGDFGSIIPGGVVGPGGVG，
SEQ ID NO.7：FFFFFRGDGSIIPGGVVGPGGVG，
SEQ ID NO.8：FFFFFGRGDSIIPGGVVGPGGVG，
SEQ ID NO.9：FFFFFGSRGDIIPGGVVGPGGVG，
SEQ ID NO.10：FFFFFGSIRGDIPGGVVGPGGVG，
SEQ ID NO.11：FFFFFGSIIRGDPGGVVGPGGVG，
SEQ ID NO.12：FFFFFGSIIPGGVRGDVGPGGVG，
SEQ ID NO.13：FFFFFGSIIPGGVVRGDGPGGVG，
SEQ ID NO.14：FFFFFGSIIPGGVVGRGDPGGVG，
SEQ ID NO.15：FFFFFGSIIPGGVVGPRGDGGVG，
SEQ ID NO.16：FFFFFGSIIPGGVVGPGRGDGVG，
SEQ ID NO.17：FFFFFGSIIPGGVVGPGGRGDVG，
SEQ ID NO.18：FFFFFGSIIPGGVVGPGGVRGDG，
SEQ ID NO.19：FFFFFGSIIPGGVVGPGGVGRGD。
in the preferred embodiment and the embodiment provided by the invention, the amino acid sequence of the polypeptide is: SEQ ID NO.19: FFFFFGSIIPGGVVGPGGVGRGD.
The source of the polypeptide is not particularly limited in the present invention, and the polypeptide can be synthesized from a conventional source in the art, for example, by using a solid-phase peptide synthesis technique.
Preferably, the mass percentage of the polypeptide in the hydrogel scaffold is 0.1-1 wt%.
Alternatively, the mass percent of the polypeptide in the hydrogel scaffold is 0.1wt%, 0.2wt%, 0.3wt%, 0.4wt%, 0.5wt%, 0.6wt%, 0.7wt%, 0.9wt%, 1.0wt%, or a value within a range of any two values.
Preferably, the mass percentage of the polypeptide in the hydrogel scaffold is 0.2wt% to 0.4wt%, or 0.5wt% to 0.7wt%. When the mass percentage of the polypeptide in the hydrogel scaffold is 0.2-0.4 wt%, the proliferation of cells is more facilitated. And when the mass percentage of the polypeptide in the hydrogel scaffold is 0.5wt% to 0.7wt%, the differentiation of cells is more facilitated.
The research of the invention finds that when the concentration of the polypeptide in the hydrogel scaffold is low, cells can proliferate in a large amount in the scaffold, and when the concentration of the polypeptide in the hydrogel scaffold is increased, the cells can start to differentiate. The hydrogel scaffold provided by the invention can be used as a good in-vitro large-scale culture scaffold for myoblasts, can be applied differently by adjusting the concentration of the hydrogel, and can be used for commercial production of cultured meat in the future.
Further, the hydrogel scaffold of the present invention includes a thickening agent.
The specific type and source of the thickener are not particularly limited, and the thickener which is nontoxic and has better biocompatibility and is suitable for cell culture addition in the field can be used.
Preferably, the thickener is selected from at least one of collagen, gelatin, matrigel, sodium alginate or nanocellulose.
Preferably, the thickening agent is collagen; the mass percentage of the thickening agent in the hydrogel bracket is 0-60 wt%.
Preferably, the animal cells are selected from chicken and/or pigeons.
Preferably, the animal cells are myoblasts.
In a second aspect, the present invention provides a method of preparing cell culture meat comprising the steps of:
s1, preparing a hydrogel scaffold, wherein the hydrogel scaffold comprises polypeptide, thickener and water; the polypeptide is obtained by inserting an RGD sequence into an amino acid sequence shown in SEQ ID NO. 1; the mass percentage of the polypeptide in the hydrogel bracket is 0.1-1 wt%;
s2, culturing animal cells by using the hydrogel scaffold prepared in the step S1.
Preferably, the temperature of the culture in step S2 is 37-41℃and the inoculum size is 0.5-1.5X10 5 individual/mL;
preferably, 4-6ng/mL of bFGF (basic fibroblast growth factor) and 8-12ng/mL of IGF-1 (insulin-like growth factor-1) are also added to the hydrogel scaffold during the culturing process of step S2. More preferably, bFGF is added at a concentration of 5ng/mL and IGF-1 is added at a concentration of 10ng/mL.
The method for preparing the cell culture meat provided by the invention utilizes the hydrogel scaffold to culture and obtain the cell culture meat. Wherein, myoblasts can not only proliferate rapidly in the hydrogel stent system, but also promote tissue regeneration and differentiation according to the change of polypeptide concentration. Through lipidomic analysis, the chicken-derived cell culture meat prepared by the method contains 297 lipids, wherein Cer lipids 3, MG lipids 4, DG lipids 52, TG lipids 102, LPC lipids 32, LPE lipids 2, LSM lipids 1, PC lipids 59, PE lipids 20, PS lipids 2 and SM lipids 20; myoblasts have a significant content change in total 69 lipid substances during meat differentiation into cells, of which 26 lipid substances with elevated content, mainly PE and TAG lipids, and 43 lipid substances with reduced content, mainly PC and DG lipids.
The method provided by the invention uses the hydrogel bracket as the cell culture carrier, has low cost, does not need to separate the hydrogel bracket in the process of culturing cells to obtain the food meat, has simple operation, and is suitable for engineering production.
The beneficial effects are that:
the invention provides an application of a hydrogel scaffold in preparing cell culture meat, wherein the hydrogel scaffold comprises polypeptide, thickener and water; the polypeptide is obtained by inserting an RGD sequence into an amino acid sequence shown in SEQ ID NO. 1. The polypeptide forms a nanofiber network structure in a hydrogel system, and is used as a bracket for cell culture, so that cells can proliferate in a large amount in the bracket. Meanwhile, with the adjustment of the concentration of the polypeptide in the hydrogel, cells can be differentiated into tissue cell forms close to slaughter meat, and the obtained cell culture meat has balanced nutrition components and high commercial value.
Drawings
In order to more clearly illustrate the technical solutions of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be described below.
FIG. 1 is a photograph of hydrogel scaffolds of different concentrations in the examples of the present invention (A, macroscopic photograph of hydrogel containing 0.1wt% polypeptide, B, macroscopic photograph of hydrogel containing 0.3wt% polypeptide, C, macroscopic photograph of hydrogel containing 0.6wt% polypeptide, D, microscopic FESEM photograph of hydrogel containing 0.1wt% polypeptide, E, microscopic FESEM photograph of hydrogel containing 0.3wt% polypeptide, F, microscopic FESEM photograph of hydrogel containing 0.6wt% polypeptide).
FIG. 2 shows the results of rheological properties of hydrogel scaffolds of different concentrations according to the present invention (the right side shows the moduli of hydrogel scaffolds in the time-based scanning-rheology mode for polypeptide concentrations of 0.1wt%,0.3wt% and 0.6wt% in order from top to bottom; and the left side shows the moduli of hydrogel scaffolds in the dynamic frequency-rheology mode for polypeptide concentrations of 0.1wt%,0.3wt% and 0.6wt% in order from top to bottom).
FIG. 3 is an infrared spectrum of hydrogel scaffolds of different concentrations according to an embodiment of the present invention.
FIG. 4 shows morphological observations before and after myoblasts of primary chicken myoblasts were myoblasted in the examples of the present invention (A, D shows morphology of nuclei of chicken myoblasts in a proliferated and differentiated state stained with DAPI, B, E shows morphology of F-actin of chicken myoblasts in a proliferated and differentiated state stained with FITC Phillidine, respectively; C, F shows combined images of morphology of nuclei of chicken myoblasts and F-actin, respectively. Scale bars are 100 μm).
FIG. 5 shows the growth of cells on hydrogels of different concentrations in the examples of the present invention (0.1 wt% hydrogel scaffolds for a1, a2, a3 and a4, 0.3wt% hydrogel scaffolds for b1, b2, b3 and b4, 0.6wt% hydrogel scaffolds for c1, c2, c3 and c 4. The incubation times for a1, b1 and c1 were 24h, the incubation times for a2, b2 and c2 were 48 h, the incubation times for a3, b3 and c3 were 96 h, the incubation times for a4, b4 and c4 were 120 h. The scale length in the figure was 100. Mu.m).
FIG. 6 shows the proliferation and nutrient consumption curves of chicken myoblasts in hydrogels with different concentrations (A, proliferation curves of chicken myoblasts; variance analysis of proliferation results of chicken myoblasts; C, concentration change of glucose in culture medium of chicken myoblasts; D, concentration change of glutamine Gln in culture medium of chicken myoblasts; E, concentration change of arginine Arg in culture medium of chicken myoblasts; F, concentration change of serine Ser in culture medium of chicken myoblasts; G, concentration change of methionine Met in culture medium of chicken myoblasts; H, concentration change of isoleucine Leu in culture medium of chicken myoblasts) according to the embodiment of the invention.
FIG. 7 shows the differentiation of PCMSC in 0.6wt% concentration polypeptide hydrogel and the characteristic markers before and after differentiation in the examples of the present invention (A, growth morphology of chicken myoblasts after 24h, 72 h and 120h, B, relative expression levels of Pax7, MYOD and MYOG mRNA in chicken myoblasts after 24h, 72 h and 120h, expression levels of MYHC protein in chicken myoblasts after 24h, 72 h and 120h, glucose, glutamine Gln, glutamic acid Glu absorption and lactic acid secretion before and after chicken myoblast differentiation, E, absorption of leucine Leu, isoleucine Ile and valine Val before and after chicken myoblast differentiation, and the scale of the expression levels of the MYHC protein in chicken myoblasts is 100 μm).
FIG. 8 is an S-plot of lipid molecules before and after myoblast differentiation of chicken myoblasts in the examples of the present invention.
FIG. 9 is a map of Heat map of differential lipid molecules before and after myoblast differentiation in chicken myoblasts in the examples of the present invention.
Detailed Description
The in-vitro large-scale amplification culture of seed cells is a key technical link for realizing the large-scale production of cell culture meat, and the in-vitro culture of the seed cells for cell culture meat production is required in an adherence culture mode at present, so that the exploration of an edible and efficient novel myoblast in-vitro amplification form is important for solving the problem of cell scale amplification.
The polypeptide hydrogel has the advantages of high water content, adjustable mechanical stability, rapid self-healing capacity, good biocompatibility, proper injectability, low toxicity and the like, and has been widely applied to different biomedical fields such as drug delivery, protein separation, biosensors, tissue engineering, wound healing and the like. With the rapid development of biological scaffold materials, the design of peptide sequences is increasingly viable. Compared with other biological extracts based on animal cells and other hydrogels produced based on crosslinking, the hydrogel has complete biocompatibility, does not contain any animal-derived components, crosslinking agents toxic to cells and the like, can form a network structure in a culture system through self-assembly, and improves the biocompatibility through covalent embedding of cell adhesion sequences such as RGD and the like, thereby creating a microenvironment similar to cells in tissues in vitro. Therefore, the material has extremely high application prospect in the aspect of in-vitro large-scale culture of the cells for culturing meat by creating a good 3D growth environment for the cells.
The invention provides an application of a polypeptide hydrogel scaffold in preparation of cell culture meat. According to the invention, through researching rheological property tests, in-vitro three-dimensional culture verification and biocompatibility analysis of polypeptide hydrogel solutions with different concentrations, it is found that cells can be subjected to three-dimensional culture in hydrogel with the concentration of 0.1wt%, but in low-concentration solution, the cells are spherically suspended and grow in a bracket; when the concentration reaches 0.3wt%, myoblasts adhere to the fibers of the hydrogel, stretch and proliferate; when the concentration is 0.6wt%, the hydrogel concentration is higher, the adhesion growth of cells is promoted, the outward extension of the cells is inhibited to a certain extent, myoblasts can be gradually fused in a bracket, the differentiation trend is presented, and the problem that the culture medium cannot be replaced can occur in a solution with higher concentration. The invention further finds that the polypeptide hydrogel is most suitable for cell growth and proliferation at the concentration of 0.3wt% by comparing the proliferation rates of cells grown in solutions with different concentrations, and can be applied to the in-vitro large-scale culture of muscle stem cells. According to the invention, through further exploration of myogenic related genes in chicken myoblasts in a higher-concentration bracket, the expression quantity of genes PAX7 and MYOD is gradually reduced along with the time increase, and the expression of a final expression gene MYOG for myodifferentiation is firstly increased and then reduced, which indicates that the chicken myoblasts gradually lose the potential for myogenic differentiation in the higher-concentration bracket, the myoblasts gradually show differentiation trend, and microscopic observation results show that the cells gradually form myotubes and possibly further form tissues. Experiments prove that the polypeptide hydrogel provided by the invention is suitable for in vitro three-dimensional culture of cells when the concentration is 0.3wt% or more. By adjusting the concentration of the hydrogel, different concentrations are suitable for different cell growth states. Higher concentrations of hydrogels are more suitable for cell growth differentiation.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention. Unless otherwise indicated, all the experimental procedures used in the examples were conventional; the materials, reagents and the like used are all commercially available.
Materials used in the following examples include:
fetal bovine serum (10099141C) was purchased from the company autothermal electric company; DMEM (H) (12100), CCK8 (CA 1210) was purchased from beijing solibao technologies limited.
Example 1
The embodiment provides an experimental method and results of culturing chicken myoblasts by using hydrogel scaffolds with different concentrations, and the experimental method and results are specifically as follows:
experimental methods
1.1 Cell extraction and culture
Separating muscle cells from leg muscle of 18-day-old chick embryo by enzyme digestion and gradient adherence (JOO S T, CHOI J S, HUR S J, et al A Comparative Study on the Taste Characteristics of Satellite Cell Cultured Meat Derived from Chicken and Cattle Muscles [ J ]]Food science of animal resources, 2022, 42 (1): 175-85.). Selecting 18-day-old chick embryo, removing muscle tissue blocks of chicken leg, removing fat composition and connective tissue, and spraying 70% alcohol for sterilization. After shearing the muscle tissue with an ophthalmic scissors, 5 volumes of 0.25% pancreatin-EDTA solution (Solarbio, T1300) were added and then subjected to enzymatic hydrolysis at 37 ℃ for 30 min. After the enzymolysis is finished, 300g of the cell mass after enzymolysis is obtained by a centrifugal machine after 5 min. After resuspension of the cells with DMEM medium (Solarbio, 11995) containing 10% FBS (Gibco, 10099141), filtration with a 100 μm sieve (Corning, 352360) was followed by direct inoculation of the cell-containing filtrate into a petri dish, which was then placed at 37℃with 5% CO 2 Is provided. After 1h of culture, the fibroblasts adhere to the walls earlier than the myoblasts, so that the supernatant is collected, 300g of the supernatant is centrifuged for 5min to obtain a cell mass, 10% FBS culture medium is resuspended and then re-inoculated into a culture dish, and when the cultured cells occupy 80% of the bottom of the dish, the cells are subjected to pancreatin digestion for passage.
1.2 Immunofluorescent staining
When the cells are cultured until the growth and the confluence of the cells at the bottom of the culture dish reach 80%, the original culture medium is sucked out, after the bottom of the culture dish is rinsed once by DPBS, the culture medium containing 2% horse serum is added, and myoblast differentiation is induced. The cell growth morphology was observed, and the cells before and after differentiation were stained for nuclei and cytoskeleton, and after the cells were washed with DPBS, 4% paraformaldehyde solution was added, and the cells were fixed at room temperature for 15 minutes, and then washed with DPBS. The DPBS solution containing 0.5% Triton X was used for 15 minutes of permeation, followed by DPBS washing. Then, the sample was stained with FITC-labeled phalloidin and Hoechst 33342 staining solution for 30 minutes at room temperature, and finally observed with a microscope and photographed.
1.3 Scanning electron microscope analysis
Samples of each concentration were deposited as 10 μl droplets onto copper plates and dried in air, and a nanofiber network of scaffolds with concentrations of 0.1wt%,0.3wt%,0.6wt% was observed under a field emission scanning electron microscope (mira 3 TESCAN).
1.4 Analysis of rheological Properties
Rheological shear measurements were performed on hydrogels of different concentrations (0.1 wt%,0.3wt% and 0.6 wt%) at 37 ℃ using a rheometer equipped with 20 mm parallel plates. The hydrogel solutions were subjected to a stabilization frequency of 1 Hz, breaking the hydrogel at 100% stress, followed by a stabilization shear strain of 1% stress for 30min, collecting their storage and loss moduli (G' and G ").
1.5 Fourier transform infrared spectroscopy
After drying the sample, the sample was ground thoroughly with KBr powder in a mortar to a mixed powder, which was pressed into nearly transparent flakes with a tablet press and tested on-press using a Fourier infrared spectrometer. Analysis of surface functional groups was performed on scaffolds of different concentrations.
1.6 Cell morphology observations
Cells were seeded in 0.1wt%,0.3wt% and 0.6wt% hydrogels in 96 well plates, respectively, and the growth of cells on the hydrogel scaffolds was observed using an optical microscope to explore the support properties of the hydrogels at different concentrations on the cells, and the three-dimensional growth morphology of the cells in the scaffolds.
1.7 Cell viability assay
200 μl of 0.1wt%,0.3wt% and 0.6wt% hydrogel solutions were added to 96-well plates, respectively, and 20000 cells were seeded per well and 3 replicates were performed. The proliferation rate of cells in the hydrogel scaffolds was measured using the CCK-8 kit. The first time was measured 0h after cell inoculation, followed by every 24h until 120h, each time absorbance was recorded at 450nm by a microplate reader.
1.8 RT-PCR analysis
After the myoblasts were inoculated into the hydrogel scaffolds for culture, the cells in the hydrogel at a concentration of 0.6wt% were collected by centrifugation. After centrifugation, a cell pellet was obtained, which was washed once with PBS, the medium and hydrogel were removed as much as possible, and then RNA was extracted by an RNA extraction kit (QIGEN, 73404) and reverse transcribed into cDNA by a cDNA reverse transcription kit (Takara, RR 037Q). The resulting cDNA was used for quantitative real-time PCR (RT-PCR) using a sequence specific primer pair of GAPDH, PAX7, MYOD, MYOG (Table 1). PCR amplification was performed by a real-time fluorescent quantitative PCR instrument (Roche, lightCycler 480 II).
TABLE 1 real-time PCR primer sequences
1.9 Glucose and lactate analysis
HPLC analysis of glucose was performed using an agilent 1260 affinity instrument equipped with an ELSD detector. The column was a Shim-pack GIST NH2 (4.6x250 mm,5 μm) with a total flow rate of 1mL/min, mobile phase a was acetonitrile and mobile phase B was water (table 2). Analysis was performed in the OpenLAB CDS, the results were expressed as absolute concentrations and corrected by standard curves as endogenous controls.
TABLE 2 Mobile phase conditions during glucose analysis
1.10 Lactic acid analysis
HPLC analysis of lactic acid was performed using Shimadzu LC-20A equipped with SPD-20A UV detector (210 nm). The column was Athena C18-WP (100A, 4.6X105 mm,5 μm) with a total flow rate of 1mL/min, mobile phase A was 0.1mol/L NH4H2PO4 solution, and mobile phase B was MeOH (Table 3). Analysis was performed in laboratory solutions, the results being expressed in absolute concentration and corrected by a standard curve as an endogenous control.
TABLE 3 Mobile phase conditions during lactic acid analysis
1.11 Amino acid analysis
HPLC analysis of amino acids was performed using an agilent 1260 informatity instrument equipped with 1260 DAD detector (338 nm and 262 nm). The column was Athena C18-WP (100A, 4.6X105 mm,5 μm) and the total flow rate was as shown in Table 4, mobile phase A was a 40mmol/L NaH2PO4 solution, pH was adjusted to 7.8 with NaOH, mobile phase B was MeOH: ACN: H2O=45:45:10 (V: V). Analysis was performed in the OpenLAB CDS, the results were expressed as absolute concentrations and corrected by single point curves as endogenous controls.
TABLE 4 Mobile phase conditions during amino acid analysis
1.12 Data analysis
Statistical analysis was performed using Prism software (GraphPad, v 9.5) and Origin 2023 software (Origin lab, v 2023). All experiments were repeated at least three times and the data were expressed as mean and standard error of the mean (shown as error bars). And the data were analyzed for one-way variance, P values less than 0.05 were considered statistically significant.
(II) results of experiments
1. Characterization of polypeptide hydrogels
1.1 Microstructure of microstructure
The microstructure of the hydrogels with different concentrations after freeze drying is observed by a scanning electron microscope, and the result is shown in fig. 1.
In FIG. 1, A, D is a hydrogel scaffold microstructure at a concentration of 0.1wt%, B, E is a hydrogel scaffold microstructure at a concentration of 0.3wt%, and C, F is a hydrogel scaffold microstructure at a concentration of 0.6 wt%. It can be seen that the hydrogel solution exhibits a porous, network-like, ordered, three-dimensional structure. The porous structure of the bracket is more beneficial to the attachment and growth of cells, migration and transportation of nutrient substances and metabolic wastes. A denser network is formed compared to a low concentration of 0.1wt%, a high concentration of 0.3wt% and 0.6 wt%.
1.2 Mechanical properties
Rheological tests show that the hydrogel provided by the invention has good self-repairing capability, and the result is shown in figure 2.
In FIG. 2, A, D is the mechanical properties of a hydrogel scaffold at a concentration of 0.1wt%, B, E is the mechanical properties of a hydrogel scaffold at a concentration of 0.3wt%, and C, F is the mechanical properties of a hydrogel scaffold at a concentration of 0.6 wt%. It can be seen that the hydrogel is destroyed under a sufficiently strong strain and that, after removal of the great strain, the hydrogel is able to re-gel, the storage modulus G' of the hydrogel solution gradually increases with time. The storage modulus recovery rate was slower for the 0.1wt% concentration hydrogel solution, after 600s the storage modulus of the material gradually increased and then was higher than the loss modulus, whereas the 0.3wt% and 0.6wt% concentration hydrogel solutions recovered earlier in the gel state. And the storage modulus of hydrogel solutions is different due to different concentrations, and the storage modulus of solutions with higher concentrations is higher. Shear-thinning hydrogels reduce viscosity upon application of shear, which is accomplished by a reversible crosslinking mechanism. After a strong shearing stress is applied to the gel at the beginning, the diluted hydrogels with different concentrations all show the shearing thinning result under the action of different shearing forces, the storage modulus of the hydrogel solution is lower than the loss modulus, and the hydrogel gradually shows the characteristics of flowing liquid. At this time, the stronger shear stress can destroy the gel structure, the hydrogen bond inside the material is broken, but the damage is temporary, once the strain or the stress stops acting, the intermolecular hydrogen bond is reconnected, the reassembling is realized, the storage modulus of the hydrogel gradually rises and gradually exceeds the loss modulus, and at the moment, the hydrogel is restored to the gel state from the liquid, so that the material has certain self-repairing capability. In practical application, operations such as blowing, vibrating, injecting, filtering, spraying and the like can apply huge shear strain or shear stress to the material, when the operations are performed, the peptide hydrogel can be thinned into a liquid state, and when the operations are terminated, the material is restored into hydrogel.
1.3 Functional group analysis
The surface functional groups of the hydrogel scaffold were analyzed by fourier infrared spectroscopy and the FITR profile is shown in fig. 3.
As can be seen, the hydrogel scaffold was at 3429cm -1 Shown as N-H stretching vibration at 1637cm -1 C=o stretching vibration, shown as amide I bond, at 1550cm -1 ，1402cm -1 Bending vibrations of N-H groups and stretching vibrations of C-N groups, shown here as amide II bonds, and C-N planar vibrations of amide III bonds (DAS M, R S, PRASAD K, et al EXTRACTION AND CHARACTERIZATION OF GELATIN: A FUNCTIONAL BIOPOLYMER [ J)]International Journal of Pharmacy and Pharmaceutical Sciences, 2017, 9:239.), which will illustrate that this hydrogel scaffold appears to contain amide linkages. From the description of hydrogels, it is known that materials are covalently embedded in cell viscosity sequences such as RGD. RGD is a tripeptide Arg-Gly-Asp sequence for cell adhesion and promoting cell adhesion, and has been found to be present mainly in ECM proteins and blood (MACPHERSON D, BRAM Y, PARK J, et al Peptides-based scaffolds for the culture and maintenance of primary human hepatocytes [ J)]Scientific Reports, 2021, 11 (1): 6772.). The surface functionalization of the material is realized by coupling or grafting the material and RGD peptide, and the hydrogel can also effectively provide adhesion sites for the in-vitro three-dimensional culture of muscle cells by covalent bonding of RGD sequences, thereby being beneficial to promoting cell growth.
2. Cell extraction and culture
2.1 extraction of chicken myoblasts
After the myoblasts were cultured in vitro, the cells were induced to differentiate and form myotubes by the addition of horse serum, and the results are shown in fig. 4.
The order of pictures in fig. 4 is from left to right, nuclear staining, cytoskeletal staining, and merging. A. B, C before differentiation, D, E, F after induced differentiation. It can be seen that myoblasts gradually grow on the culture dish, and after induced differentiation, part of myoblasts gradually fuse and form myotubes. This suggests that chicken myoblasts were successfully extracted and then the myoblasts could be cultured in vitro and induced to differentiate.
2.2 Culture of chicken myoblasts in polypeptide hydrogels
To verify the biocompatibility of scaffolds, cultured muscle stem cells were isolated at 1×10 5 The concentration of each mL was seeded on a hydrogel scaffold of 0.1wt%,0.3wt%,0.6wt%, and the growth state of the cells was observed daily by a microscope, and the results are shown in FIG. 5.
In FIG. 5, a1, a2, a3, a4 are 0.1wt% hydrogel scaffolds, b1, b2, b3, b4 are 0.3wt% hydrogel scaffolds, and c1, c2, c3, c4 are 0.6wt% hydrogel scaffolds. The cell growth was followed from the first, second, fourth and fifth days in this order. It can be seen that at a concentration of 0.1wt%, cells will not adhere to the bottom of the 96-well plate but will grow in a three-dimensional space where the morphology of the cells is spherical and the cells in the hydrogel will proliferate by dividing one cell into two cells. When the concentration reaches 0.3wt%, cells adhere to fibers and stretch, the cells form in the three-dimensional space when in adherent culture, and the higher the concentration, the more obvious the stretching state of the cells on the scaffold with the concentration of 0.6 wt%.
2.3 Proliferation of chicken myoblasts in polypeptide hydrogels
The proliferation rate of cells on scaffolds of different concentrations was studied by CCK-8 experiments, the results are shown in FIG. 6.
In FIG. 6, proliferation curves of chicken myoblasts; B. analysis of variance of chicken myoblast proliferation results; C. concentration change of glucose in the culture medium during the culture process of chicken myoblasts; D. concentration change of glutamine Gln in the culture medium during chicken myoblast culture; E. concentration change of arginine Arg in a culture medium during chicken myoblast culture process; F. concentration change of serine and Ser in the culture medium during the culture process of chicken myoblasts; G. methionine Met concentration change in the culture medium during the culture process of chicken myoblasts; H. concentration of isoleucine Leu in the medium was varied during chicken myoblast culture. It can be seen that the growth rate of myoblasts on hydrogel scaffolds was significantly higher than that of control groups in which cells were cultured in two dimensions, as compared to by adherent 2D culture on 96-well plates, and that the growth rate of cells on scaffolds at different concentrations also exhibited different differences. The proliferation rate of cells on 0.3wt% of scaffolds was higher compared to the 0.1wt% scaffold concentration system.
In the adherent culture, cells are attached to the surface of plastic to grow in a single layer mode, and at the moment, the cell culture cost is low, and the operation is simple and convenient. However, due to the difference in cell growth and the environment in the tissue, the morphology of the cells changes, and the cell differentiation, proliferation, viability, gene and protein expression, response to stimuli, drug metabolism and other cell functions tend to change in adverse directions (KAPA Ł CZY Ń SKA M, KORENDA T, PRZYBY Ł A W, et al 2D and 3D cell cultures-a comparison of different types of cancer cell cultures [ J ]. Arch Med Sci, 2018, 14 (4): 910-9.). The three-dimensional culture can better reduce the problems caused by the in-vitro culture of the cells because the environment of the extracellular matrix is imitated. When cells are cultured in three dimensions, some cells under non-adherent conditions exhibit reduced cell-to-cell and cell-to-matrix interactions, lose anchoring, and form cell spheres. Therefore, when the concentration is low, the hydrogel is used for providing a certain support for the cells, so that the cells cannot settle to the bottom of the culture dish when being inoculated in a liquid culture medium, but the environment is still a condition unsuitable for cell adhesion, and the cells are in the form of cell spheres in the culture medium (see a1, a2, a3 and a4 in fig. 5). With the increase of the concentration of the scaffold material and the higher content of the hydrogel, the support effect of the scaffold on the cell growth is stronger, and the scaffold material provides a certain adhesion site for the cell. At this time, the cells can adhere to the fibers in the scaffold like the adherent cells, and take on an expanded form, so that myoblasts can be seen not to be spherical any more in higher concentration but to take on a tendency to grow outward in three-dimensional space (see b1, b2, b3 and b4 in fig. 5).
Adherent cells need to adhere to a medium to grow, and they explore the environment by expanding platy and filopodia as the cells interact with the substrate. The physicochemical properties of the scaffold itself (morphology, pore size, surface morphology, etc.) can thus directly influence the behavior of the cells. The results of the proliferation rate of cells (FIG. 6, A) demonstrate that the high concentration hydrogel scaffold provides a surface for cells to attach to, and allows for a certain information exchange between cells in an extended state, and that the proliferation rate of cells is higher than that of cells in a spherical shape, i.e., scaffolds with concentrations of 0.3wt% and above are more conducive to the growth of muscle stem cells in three dimensions. However, too high a concentration of hydrogel may make the solution more viscous, which in turn may inhibit cell expansion, eventually tending to fuse and differentiate myoblasts. Therefore, we further confirmed the growth state of cells in hydrogels grown at this concentration by experiments such as gene, protein detection and metabolic analysis. Meanwhile, we also observed that myoblasts tended to differentiate when the concentration reached 0.6wt% on day 5 without the addition of differentiation medium, and muscle fibers were gradually formed (see c1, c2, c3 and c4 in fig. 5).
3. Differentiation of PCMs in polypeptide hydrogels
Results of RT-PCR assay and WB assay of MYHC protein containing relative mRNA expression levels of Pax7, myoD and MyoG in chicken myoblasts at different culture periods in 0.6wt% hydrogel system are shown in FIG. 7.
It can be seen that the marker gene PAX7 of the myosatellite cells and the myogenic differentiation early gene MYOD gradually show a decreasing trend within five days, the myogenic differentiation end gene MYOG shows a trend of increasing and then decreasing, and MYHC shows a trend of increasing expression. The PAX7 gene is a marker gene for the resting and activated state of the muscle satellite cells, and expression of the gene is drastically reduced before differentiation of the muscle satellite cells (OLGUIN H C, OLWIN B. Pax-7 up-regulation inhibits myogenesis and cell cycle progression in satellite cells: a potential mechanism for self-renewal [ J ]. Dev Biol, 2004, 275 (2): 375-88.). MYOD plays a major role in myogenic differentiation, inducing differentiation of pre-myoblasts into myoblasts; MYOG plays a role in the terminal differentiation of myocytes, maturation and maintenance of myofibers, and is generally considered a key factor in terminal myocyte differentiation (Wang Nan, li Jianfeng, wang Lixiao, et al Pax7, myoD and MYOG genes for preliminary studies to infer time to injury [ J ]). Down-regulation of PAX7 and MYOD genes indicated that cells grown at 0.6wt% concentration were gradually losing the stem properties of myosatellite cells, while the first high and then low expression of MYOG indicated that cells were differentiating in the direction of myoblasts. This further demonstrates that high concentrations of hydrogel scaffolds can promote progressive differentiation of myoblasts during three-dimensional growth. The high expression of MYHC on the third day of culture demonstrated the production of the myosin heavy chain, an important marker in muscle, demonstrating the completion of myogenic differentiation of chicken myoblasts. Meanwhile, from the metabolic analysis results, it can be seen that the absorption rates of glucose and three amino acids by chicken myoblasts before and after differentiation are significantly different.
Therefore, the invention provides an application of a hydrogel scaffold in preparing cell culture meat, and a good effect is obtained in research on preparing the cell culture meat by exploring the composition of polypeptide hydrogel and trying to use the hydrogel scaffold as an in vitro 3D culture scaffold of myoblasts. The invention researches the physical property, microcosmic appearance, rheological property and surface functional group of the polypeptide hydrogel, and discovers that the hydrogel has a porous three-dimensional structure and good self-repairing property after being damaged by external force, and the surface functional group mainly shows characteristic peaks of amide bonds. Furthermore, the invention selects the brackets with different concentrations for the in-vitro three-dimensional culture of the chicken myoblasts, and compared with the adherent culture, the three-dimensional culture efficiency of the cells on the brackets is generally higher than that of the two-dimensional culture. However, the change of the concentration of the scaffold can influence the growth form of cells, the cells are spherical in the scaffold with low concentration, and after the concentration is increased, myoblasts can stretch and grow on the scaffold to form a fusiform shape. Analysis of relevant signal molecules inside chicken myoblasts shows that the cell myoblasts related genes PAX7 and MYOD have a downward regulation trend along with the time increase, the expression of the gene MYOG is firstly increased and then decreased, and the expression of MYHC protein is suddenly increased after three days of culture, so that the chicken myoblasts are gradually differentiating in a high-concentration bracket.
The research result of the invention not only shows that the polypeptide hydrogel scaffold material has good potential of being applied to the in-vitro proliferation of chicken myoblasts, but also has remarkable effect of regulating and controlling the directional differentiation of chicken myoblasts.
Example 2
The embodiment provides a detection method and an analysis result of lipid substance nutrient content before and after differentiation of chicken myoblasts, which are specifically as follows:
experimental methods
1. Extraction of esters
Each sample was taken at 2X 10 6 Adding 10 mu L of internal standard solution and 490 mu L of methanol solution (preparation of the methanol solution: adding 630 mg ammonium formate and 1mL formic acid into a 1L volumetric flask, fixing the volume by methanol), performing ultrasonic crushing, performing vortex for 1 min, centrifuging (10000 r/min, 4 ℃) for 5min in a low-temperature centrifuge, and collecting supernatant and uploading.
2. Mass spectrometry of lipids
Scan type: full MS- -dd-MS 2, ion mode: positive, sheath gas flow rate 40 L.min-1, auxiliary gas flow rate 15 L.min-1, spray voltage 3.5. 3.5 KV, capillary temperature 325 ℃, auxiliary gas heater temperature 450 ℃, full MS resolution 60000, dd-MS2 resolution 15000.
3. Database search and peak area confirmation of lipids
The lipid molecular ion identification and the data alignment analysis of the cell samples before and after differentiation are carried out on the non-targeted lipid identification through a LipidSearch software product ion search and annotation alignment algorithm, and the peak areas of the molecular ion peaks of the corresponding lipid compounds are derived after the ion identification is carried out one by one manually.
4. Quantitative analysis of lipids
The lipid is quantified by taking TG (17:0/17:1/17:0) -d5, PC (17:0/14:1) -d5 and PE (17:0/16:1) -d5 as internal standard substances, and performing quantitative analysis and calculation on other lipid compounds by an internal standard method.
5. Statistical analysis of lipids
The statistical analysis of lipid is based on quantitative and qualitative analysis of lipid, S-plot curves before and after differentiation of chicken myoblasts are obtained by utilizing SIMCA (V14.1) software firstly through OPLS-DA analysis, then lipid compounds with VIP >1 and P <0.05 are screened out from the S-plot curves as differential substances before and after differentiation, concentration data of the differential substances before and after differentiation of chicken myoblasts are further imported into MetaboAnalyst (V5.0) for Statistical Analysis [ one factor ] analysis, and finally, a lipid heat map with significant difference before and after differentiation of chicken myoblasts is obtained through analysis.
(II) results of experiments
1. Qualitative analysis of lipids
Through the search of the LipidSearch database and the manual confirmation of the primary parent ion and secondary child ion structures, 297 lipid substances are obtained through the co-analysis and identification of chicken-derived cell culture meat in the embodiment, wherein Cer lipid 3, MG lipid 4, DG lipid 52, TG lipid 102, LPC lipid 32, LPE lipid 2, LSM lipid 1, PC lipid 59, PE lipid 20, PS lipid 2 and SM lipid 20.
2. Quantitative analysis of lipids and characterization of lipids
After lipid quantification, the results are shown in fig. 8 by analysis of large data on up to 297 lipid species. As can be seen from FIG. 8, the levels of various lipid components in the myoblasts were shifted up or down, indicating that the levels of lipid-based nutrients were significantly changed before and after the differentiation of the chicken myoblasts. As shown in table 5: there are 69 lipid compounds in which significant content changes (VIP >1, P < 0.05) occurred.
TABLE 5 characteristic lipid molecules of chicken myoblasts before and after myogenic differentiation
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3. Analysis of characteristic lipid content
The results of analysis of the content change of 69 characteristic lipids with significant differences are shown in fig. 9.
From the figure, it can be seen that the myoblasts had a significant change in the content of 69 lipid substances during meat differentiation into cells, 26 lipid substances with increased content, mainly PE and TAG lipids, and 43 lipid substances with decreased content, mainly PC and DG lipids. The total lipid content of chicken myoblasts before and after myogenic differentiation is shown in Table 6.
TABLE 6 lipid content of chicken myoblasts before and after myogenic differentiation
Among the alternative protein foods that are currently common, cell-cultured meat is the only alternative meat food product that is produced based on animal cells. Cell culture meats have the basis of fully retaining the nutritional components of animal derived products compared to vegetable protein-based and microbial protein-based alternative protein-based foods. Through the research of the embodiment, the chicken-derived cell culture meat prepared by the method has rich lipid nutrient components, the total variety can reach 297 varieties, meanwhile, 69 kinds of lipids have obvious content change in the process of differentiating and developing the cell culture meat, which also shows that the lipid nutrient components are continuously changed in the process of preparing the cell culture meat, and the invention also provides a basis for the subsequent technical development of regulating the content of the lipid nutrient components in the cell culture meat by an artificial method.
The above examples merely represent a few embodiments of the present invention, which facilitate a specific and detailed understanding of the technical solutions of the present invention, but are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention.

Claims (10)

1. The application of the hydrogel scaffold in preparing cell culture meat is characterized in that the hydrogel scaffold is used for culturing animal cells; the hydrogel scaffold comprises a polypeptide, a thickener and water; the polypeptide is obtained by inserting an RGD sequence into an amino acid sequence shown in SEQ ID NO. 1.
2. The use according to claim 1, wherein the amino acid sequence of the polypeptide is selected from at least one of the sequences shown in SEQ ID No.2 to 19.
3. The use according to claim 1, wherein the mass percentage of the polypeptide in the hydrogel scaffold is 0.1-1 wt%.
4. The use according to claim 3, wherein the mass percentage of the polypeptide in the hydrogel scaffold is 0.2wt% to 0.4wt%, or 0.5wt% to 0.7wt%.
5. The use according to any one of claims 1 to 4, wherein the thickener is selected from at least one of collagen, gelatin, matrigel, sodium alginate or nanocellulose.
6. The use according to claim 5, wherein the thickening agent is collagen; the mass percentage of the thickening agent in the hydrogel bracket is 0-60 wt%.
7. The use according to any one of claims 1 to 4, wherein the animal cells are selected from chicken and/or pigeons.
8. The use according to claim 7, wherein the animal cells are myoblasts.
9. A method for preparing cell culture meat, comprising the steps of:
s1, preparing a hydrogel scaffold, wherein the hydrogel scaffold comprises polypeptide, thickener and water; the polypeptide is obtained by inserting an RGD sequence into an amino acid sequence shown in SEQ ID NO. 1; the mass percentage of the polypeptide in the hydrogel bracket is 0.1-1 wt%;
s2, culturing animal cells by using the hydrogel scaffold prepared in the step S1.
10. The method according to claim 9, wherein the temperature of the culture in step S2 is 37-41℃and the inoculum size is 0.5-1.5X10) 5 individual/mL; the hydrogel bracket is also added with 4-6ng/mL of bFGF and 8-12ng/mL of IGF-1.
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