CN109136186B - Suspension culture method of human skull mesenchymal stem cell-derived neuron-like cells - Google Patents

Suspension culture method of human skull mesenchymal stem cell-derived neuron-like cells Download PDF

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CN109136186B
CN109136186B CN201811148051.3A CN201811148051A CN109136186B CN 109136186 B CN109136186 B CN 109136186B CN 201811148051 A CN201811148051 A CN 201811148051A CN 109136186 B CN109136186 B CN 109136186B
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卢刚
麻育源
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Zhejiang Provincial Peoples Hospital
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Abstract

The invention relates to a suspension culture method of neuron-like cells derived from human skull mesenchymal stem cells, which comprises the following steps: extracting bone marrow mesenchymal stem cells from human skull fragments or fragments as tissue source, culturing and incubating with the amount of 104～106Per cm2Inoculating to the inducing liquid; selecting finished gellan gum, heating to above 90 ℃ to prepare a solution with a mass fraction ratio of 1-3% for later use; observing the density and the pore size of a network structure in the suspension gel prepared from the gellan gum solutions with different concentrations by using an electron microscope or a laser confocal microscope; and (4) transferring the selected neural stem cells into a suspension gel-DMEM medium to complete culture proliferation. The invention has the beneficial effects that: the method of the invention makes up the technical defects of the existing laboratory conditions and the restriction of objective factors such as gravity and the like, combines the biological behavior and functional morphological characteristics of the neural stem cells, and can effectively induce and differentiate the human skull mesenchymal stem cells by preparing the suspension gel-DMEM culture medium by utilizing the modified gellan gum.

Description

Suspension culture method of human skull mesenchymal stem cell-derived neuron-like cells
Technical Field
The invention relates to the technical field of cell culture, in particular to a suspension culture method of human skull mesenchymal stem cell-derived neuron-like cells.
Background
Craniocerebral trauma, cerebrovascular accidents and spinal cord injury are common diseases and frequently encountered diseases worldwide and are also the leading factors causing disability after the injury. Despite the great advances made in the emergency treatment and early rehabilitation of craniocerebral trauma and cerebrovascular accidents, there is still a lack of effective treatment for neurological deficits in the latter stages of craniocerebral trauma and cerebrovascular accidents. Cell transplantation provides a new direction for the treatment of the diseases, but because nerve cells used for cell transplantation cannot be obtained and exogenous nerve cells are difficult to culture and expand, finding a culture method which can construct a perfect three-dimensional tissue carrier for the nerve stem cells under in vitro conditions to maintain the differentiation and proliferation of the cells and promote the regeneration of tissues and exert functions in the transplanted defective tissues becomes the key of the treatment. Gellan gum (gellan gum) is an anionic linear exopolysaccharide produced by gram-negative aerobic bacillus (sphingomonas paucimobilis), and with the development of suspension gel technology, gellan gum has attracted much attention in the medical field due to its excellent gelling properties and ion sensitivity, and is mainly used as a carrier for cell transplantation, particularly in the fields of pharmaceutics and tissue engineering. Under the conventional conditions, cells cannot be distributed to form a three-dimensional tissue structure due to the action of gravity in the culture or transplantation of the neural stem cells, and the suspension gel prepared by utilizing the modified gellan gum can not only construct a proper three-dimensional tissue carrier for the induced differentiation, culture and proliferation of the neural stem cells under the in vitro condition, but also provide a tissue framework for promoting the connection regeneration and playing the functions of the neural cells for the cell transplantation treatment of the central nervous system in the future. The suspension culture method of the neuron-like cells derived from the human skull mesenchymal stem cells, provided by the invention, gives consideration to experimental research and clinical application of the neural stem cells, and provides a new means and method for differentiation and proliferation of the neural stem cells and clinical stem cell transplantation treatment under laboratory conditions in the future.
Disclosure of Invention
The invention aims to overcome the defects and provide a suspension culture method of neuron-like cells derived from human skull mesenchymal stem cells.
A suspension culture method of neuron-like cells derived from human skull mesenchymal stem cells comprises the following steps:
step one, extracting bone marrow mesenchymal stem cells by using human skull fragments or fragments as tissue sources, culturing and incubating the bone marrow mesenchymal stem cells by 104～106Per cm2Is inoculated into an inducing solution, and the composition of the inducing solution is as follows: 1 XN 2, 1 XB 27, 30-50 ng/ml B-FGF, DMEM/F12 basic culture solution as solvent, and CO concentration at 37 ℃/5%2Differentiating, inducing and culturing for 21 days in an incubator to obtain neuron-like cells;
step two, selecting finished gellan gum, heating to above 90 ℃ to prepare a solution with a mass fraction ratio of 1-3% for later use, wherein the solvent is PBS, and inoculating according to cell cultureDensity and requirement for inducing differentiation, adding prepared gellan gum solution into DMEM medium and adding into the DMEM medium at concentration of 10mmol/dm3Cooling to 30-35 deg.C to obtain suspension gel;
thirdly, observing the density and the pore size of the network structure in the suspension gel prepared by the gellan gum solution with different concentrations by adopting an electron microscope or a laser confocal microscope, and screening out the proportion concentration of the suspension gel suitable for neuron-like cell culture proliferation and induced differentiation and a corresponding carrier structure;
and step four, transferring the selected neural stem cells into a suspension gel-DMEM medium to complete culture proliferation.
Preferably, the method comprises the following steps: the final concentration composition of the suspension gel in the second step: 0.5-1.5 percent of gellan gum with the mass fraction ratio of 10mmol/dm3Calcium chloride solution, solvent DMEM/F12 basic culture solution.
Preferably, the method comprises the following steps: in the fourth step, the gellan gum with the concentration of 0.01-0.1% by mass percent is used for preparing a suspension gel DMEM medium.
Preferably, the method comprises the following steps: in the third step, synapses of the screened neuron-like cells extend, the cells shrink to be in a similar circle shape, synapses of adjacent cells are connected, and the examination of an ultra-micro morphological structure proves that intermediate connection is formed among the neuron-like cells and villous and synapse-like structures exist.
The method has the advantages that the method makes up the technical defects of the existing laboratory conditions and the restriction of objective factors such as gravity and the like, combines the biological behavior and functional morphological characteristics of the neural stem cells, utilizes the modified gellan gum to prepare the suspension gel-DMEM culture medium, can effectively induce and differentiate the human skull mesenchymal stem cells, and provides a proper three-dimensional tissue carrier for the growth and functional remodeling of the neuron-like cells, and more importantly, the differentiated and mature neuron-like cells not only have the morphological characteristics of the neural cells, but also have the formation of functional microvilli and synapse-like structures and the establishment of intercellular intermediate connection.
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FIG. 1 is a morphological micrograph of human skull mesenchymal stem cell-derived neuron-like cells illustrating synaptic elongation, soma shrinkage and connection of adjacent cell synapses. Magnification is 100 times.
FIG. 2 is a morphological micrograph of human cranial mesenchymal stem cell-derived neuron-like cells illustrating serial proliferative growth of the neuron-like cells in suspension gel-DMEM medium. Magnification is 100 times.
FIG. 3 is a NeuN and NSE double-labeled immunofluorescence assay of human skull mesenchymal stem cell-derived neuron-like cells, and the result shows positive. Magnification is 100 times.
FIG. 4 is a schematic diagram showing the network structure of a suspension gel prepared from a gellan gum solution with a mass fraction ratio concentration of 0.06% on the left and 0.04% on the right under a confocal laser scanning microscope;
FIG. 5 is a structural diagram of ultramicromorphism of human skull mesenchymal stem cell-derived neuron-like cells cultured and proliferated in a suspension gel-DMEM medium under an electron microscope, illustrating microvilli and synapse-like structures on the cell body surface.
FIG. 6 is a diagram showing the structure of ultramicromorphism of human skull mesenchymal stem cell-derived neuron-like cells cultured and proliferated in a suspension gel-DMEM medium under an electron microscope, illustrating the intermediate junctions between the neuron-like cells.
Detailed Description
The present invention will be further described with reference to the following examples. The following examples are set forth merely to aid in the understanding of the invention. It should be noted that, for those skilled in the art, it is possible to make various improvements and modifications to the present invention without departing from the principle of the present invention, and those improvements and modifications also fall within the scope of the claims of the present invention.
Example 1: isolation of human skull mesenchymal stem cells
Is divided into 10cm3Cutting skull bone pieces into pieces by using a medical electric drill saw, transferring the cut pieces into a 50mL centrifuge tube, adding 25mL physiological saline, uniformly mixing, placing the cut pieces into a low-temperature shaking table, shaking for 15 minutes at the temperature of 8 ℃ and the rotating speed of 100-200 rpm, filtering by using a 200-mesh screen, centrifuging the filtrate at 1000rpm for 6min, removing supernatant, suspending the precipitate by using a cell culture solution, and taking a small amount of suspension to pass through a full-automatic centrifugeAfter counting by the hemodynamics analyzer, the count is 2X 105/cm2Inoculating to density of 25cm2And (3) placing the plastic cell culture bottle in an incubator containing 5% CO2 at 37 ℃ for culture, pouring the culture bottle after 48 hours to remove the non-adherent cells, changing a fresh culture solution, changing the solution every 2 days, digesting and passaging the cells until the cells grow to 90% confluence until the cells grow to P3 generations, and obtaining the fibrous human skull mesenchymal stem cells with single form.
Example 2: in vitro differentiation induction of human skull mesenchymal stem cells into neuron-like cells
Inoculating the P3 generation human skull mesenchymal stem cells obtained in the example 1 into inducing liquid at 37 ℃ and 5% CO2Inducing and culturing for 21 days in an incubator, wherein liquid is changed every 2 days, and collecting induced cells for identification to obtain mature neuron-like cells.
The final concentration composition of the induction liquid is as follows: 1 XN 2, 1 XB 27, 50ng/ml B-FGF in DMEM/F12 basic medium (from Corning). N2, B27 were purchased from Gibco, and B-FGF was purchased from Peprotech.
Example 3: culture proliferation of human skull mesenchymal stem cell-derived neuron-like cells
The neuron-like cells obtained in example 1 were inoculated into suspension gel-DMEM medium prepared with gellan gum of various concentrations and placed at 37 ℃/5% CO2Culturing and proliferating in an incubator conventionally, collecting cells, observing and detecting.
Preparation of suspension gel: finished gellan gum (Gelrite)@) Heating to prepare solution, adding calcium chloride solution, cooling, mixing with DMEM/F12 basic culture solution, and placing in a culture container. Finished gellan gum (Gelrite)@) Are commercially available.
Example 4: identification of human skull mesenchymal stem cell-derived neuron-like cells
(1) And (3) morphological observation: the cell morphology of the uninduced human skull mesenchymal stem cells and the cells induced to the 21 st day by the induction liquid are respectively observed by an optical microscope, and the cell morphology is photographed and recorded, and the result is shown in figure 1 and is magnified by 100 times.
(2) Immunofluorescence detection of nerve cell marker protein: after cells induced by the induction liquid are fixed for 15min at room temperature (25 ℃) by 4% paraformaldehyde, the cells are rinsed by PBS (pH value is 7.4) to remove the paraformaldehyde, then the cells are soaked and treated for 10min at room temperature by 0.05% Triton X-100 (purchased from Sigma), then 5% goat serum (purchased from biologies) is used for sealing for 1h at room temperature, the PBS is used for cleaning twice, the cells are divided into 4 groups, primary antibodies are respectively added, the group 1 is anti-Tuj-1, the group 2 is anti-NeuN and anti-NSE, the group 3 is anti-Syn, the primary antibodies are all purchased from Abcam, and the cells are incubated overnight in a wet box at 4 ℃; soaking and washing with PBS for three times, each time for 10 min; adding a fluorescence-labeled secondary antibody, wherein a group 1 is Alexa 488, a group 2 is Alexa 488 and Alexa 647-labeled secondary antibody, a group 3 is Alexa 647 (the secondary antibodies are all purchased from Unico biological company), incubating for 1h at 37 ℃, and performing PBS immersion washing for three times; DAPI (purchased from Sigma) staining nuclei and photographing were performed by laser confocal microscopy (Carl Zeiss). The results are shown in FIG. 3, at 100 times magnification.
(3) Preparation of suspension gel: finished gellan gum (Gelrite)@) Heating to 90 deg.C, adding distilled water to obtain 0.01-0.1% solution, adding 0.02%, 0.04%, 0.06%, 0.08% and 0.1% gellan gum solution, and adding 10mmol/dm3The calcium chloride solution is cooled to 30-35 ℃ at room temperature, and then DMEM/F12 basic culture solution is added to obtain the suspension gel-DMEM culture medium. The compactness and the pore size of a network structure in the suspension gel prepared by gellan gum solutions with different concentrations are observed by adopting a laser confocal microscope, the proportion concentration of the suspension gel and the corresponding carrier structure which are suitable for neuron-like cell culture proliferation and induced differentiation are screened out, and the selected nerve cells are transferred into a suspension gel-DMEM medium to complete culture proliferation.
(4) Detecting the microstructure of the nerve cells: collecting cells induced for 21 days in a centrifuge tube, centrifuging at 1000rpm/min for 10min, discarding supernatant, fixing cell precipitate with 2.5% glutaraldehyde, fixing at 4 deg.C for 30min, rinsing with PBS for 3 times, fixing with 1% osmic acid for 2h, rinsing with PBS, dehydrating with acetone, embedding, slicing at 90nm, and taking pictures by transmission electron microscope. The results are shown in FIGS. 5 and 6.
The result shows that the cultured neuron-like cells have morphological characteristics of nerve cells and expression of nerve cell marker proteins (NeuN and NSE double-labeling positive), and in addition, the neuron-like cells cultured and propagated by the suspension gel-DMEM medium have synapse-like structures and microvilli which are characteristic of the nerve cells and are formed with intercellular intermediate junctions under an electron microscope, which indicates that the suspension culture method can effectively promote the proliferation of the neuron-like cells and generate effective intercellular junctions.

Claims (2)

1. A suspension culture method of neuron-like cells derived from human skull mesenchymal stem cells is characterized by comprising the following steps:
step one, extracting bone marrow mesenchymal stem cells by using human skull fragments or fragments as tissue sources, culturing and incubating the bone marrow mesenchymal stem cells by 104～106Per cm2Is inoculated into an inducing solution, and the composition of the inducing solution is as follows: 1 XN 2, 1 XB 27, 30-50 ng/ml B-FGF, DMEM/F12 basic culture solution as solvent, and CO concentration at 37 ℃/5%2Differentiating, inducing and culturing for 21 days in an incubator to obtain neuron-like cells;
step two, selecting finished gellan gum, heating to the temperature of more than 90 ℃ to prepare a solution with the mass fraction ratio of 1-3% for later use, using PBS as a solvent, adding the prepared gellan gum solution into a DMEM medium and adding the solution with the concentration of 10mmol/dm according to the cell culture inoculation density and the requirement of induced differentiation3Cooling to 30-35 deg.C to obtain suspension gel; the final concentration of the suspension gel consisted of: 0.5-1.5 percent of gellan gum with the mass fraction ratio of 10mmol/dm3Calcium chloride solution, wherein the solvent is DMEM/F12 basic culture solution;
thirdly, observing the density and the pore size of the network structure in the suspension gel prepared by the gellan gum solution with different concentrations by adopting an electron microscope or a laser confocal microscope, and screening out the proportion concentration of the suspension gel suitable for neuron-like cell culture proliferation and induced differentiation and a corresponding carrier structure;
step four, preparing a suspension gel DMEM medium by using gellan gum with the mass fraction ratio of 0.01-0.1%; and (4) transferring the selected neural stem cells into a suspension gel-DMEM medium to complete culture proliferation.
2. The method for suspension culture of human skull mesenchymal stem cell-derived neuron-like cells according to claim 1, wherein: in the third step, synapses of the screened neuron-like cells extend, the cells shrink to be in a similar circle shape, synapses of adjacent cells are connected, and the examination of an ultra-micro morphological structure proves that intermediate connection is formed among the neuron-like cells and villous and synapse-like structures exist.
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CN105505875A (en) * 2016-02-16 2016-04-20 杨廷稳 Culture medium and culture method for efficiently inducing stem cell directional differentiation

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