1. A method for separating osteoblasts by combining enzyme digestion and tissue block culture, which is characterized by comprising the following steps:

s1, early preparation: taking skull tissues within seven days, cleaning and then shearing the skull tissues into tissue blocks;

s2, enzyme digestion: placing the tissue blocks in a centrifugal tube, and digesting the tissue blocks step by trypsin and type I collagenase until the tissue blocks are loosened into floccules;

s3, cell culture: adding culture solution into the centrifugal tube, standing, changing the culture solution in a half-liquid changing mode every 1-3 days until cells grow out from the periphery of the tissue block, changing the culture solution and discarding the tissue block;

s4, cell purification: and (4) removing the fibroblasts in the centrifuge tube in the step S3 by a differential wall pasting method to obtain the purified osteoblasts.

2. The method for separating osteoblasts by enzyme digestion in combination with tissue mass culture according to claim 1, wherein the enzyme digestion in step S2 is performed as follows:

1) respectively transferring the tissue blocks with the consistent volumes in the step S1 into a centrifugal tube, adding 0.25% trypsin solution with the volume 5-6 times that of the tissue blocks into the centrifugal tube, placing the centrifugal tube into a constant-temperature water bath kettle for oscillating digestion, blowing the tissue blocks to make the digested cells fall off, removing the upper solution after centrifugation, and cleaning with PBS solution;

2) after the cleaning is finished, adding type I collagenase which is 5-6 times of the volume of the tissue block, continuously oscillating and digesting in a constant-temperature water bath kettle, centrifuging and removing an upper layer solution;

3) repeating the step 2) until the tissue blocks are loosened into floccules.

3. The method for separating osteoblasts by enzyme digestion in combination with tissue mass culture according to claim 2, wherein in step 1), the time for shaking digestion is 20-40 min; in the step 2) and the step 3), the time of shaking digestion is 15-25 min.

4. The method for culturing and separating osteoblasts by combining enzyme digestion and tissue mass according to claim 2, wherein the temperature of the constant temperature water bath is 34-37 ℃.

5. The method for separating osteoblasts by enzyme digestion in combination with tissue mass culture according to claim 1, wherein the size of said tissue mass is 1-3mm in step S1³。

6. The method for separating osteoblasts by enzyme digestion and tissue block culture according to claim 1, wherein in step S3, the semi-liquid changing method comprises:

using a rubber-tipped dropper to suck 1ml of the cell culture solution in the centrifuge tube and discard the cell culture solution, and adding 1ml of new culture solution into the centrifuge tube.

7. The method for separating osteoblasts by enzyme digestion and tissue block culture according to claim 1, wherein in step S4, the differential adherence method comprises the following steps:

sucking old culture solution in a centrifuge tube, discarding, changing the culture solution every 24-36h, digesting with 0.25% trypsin solution, adding equal amount of culture solution to terminate digestion, and blowing and beating cells on the bottle wall and the wall corner to disperse the cells to form cell suspension;

the centrifuge tube was transferred to a six-well plate at 37 ℃ with 5% CO₂After culturing in the incubator, centrifuging the culture solution and the nonadherent cells in the centrifuge tubeSucking out, centrifuging, adding cell culture solution, suspending, transferring into a new six-well plate, and treating with 5% CO at 37 deg.C₂Culturing in an incubator to obtain the purified osteoblasts.

Background

The growth and development of bones are a dynamic balance process between bone formation and bone absorption, the osteoblasts determine the growth and development of the bones and influence the generation and formation of the bones, play a very important role in the synthesis and secretion of collagen and glycoprotein in bone matrixes, can keep the relative stability of various physicochemical factors of extracellular fluid of animal body cells, coordinate various physiological mechanisms and life activities in animal bodies, play an important role in treating various diseases or epidemic diseases caused by the bones, are mainly derived from periosteum and bone tissues in vivo, have a plurality of specimens for culturing the osteoblasts, mainly derived from human embryos, rats, mice and rabbits, use the mice as specimens which are economical, practical and not difficult to obtain, and have extremely active osteoblast activities of the skull of the suckling mice, show good proliferation capacity, and have a large number of cells collected by the skull of the newborn suckling mice, therefore, we selected suckling mice as specimen to isolate and culture osteoblasts in the experiment.

The research on osteoblasts is carried out to establish a correct osteoblast separation culture method, the research on the bone tissue metabolism has a key effect, the method is the basis of bone tissue engineering and a bone tissue metabolism formation mechanism, because osteoblasts exist in compact tissues, the osteoblasts are difficult to completely separate, the purity of the osteoblasts which are separated and cultured is different, and the method for separating and culturing osteoblasts by a simple enzyme digestion method or a simple tissue mass culture method has limited number of separated cells or low cell purity and cannot meet the requirements of experiments, so that the establishment of the method for separating and culturing osteoblasts is very important.

Disclosure of Invention

In view of the above-mentioned disadvantages of the prior art, it is an object of the present invention to provide a method for separating osteoblasts by enzyme digestion in combination with tissue mass culture.

In order to achieve the above objects or other objects, the present invention is achieved by the following technical solutions:

a method for separating osteoblasts by combining enzyme digestion and tissue block culture, S1, pre-preparation: taking skull tissues within seven days, cleaning and then shearing the skull tissues into tissue blocks;

s2, enzyme digestion: placing the tissue blocks in a centrifugal tube, and digesting the tissue blocks step by trypsin and type I collagenase until the tissue blocks are loosened into floccules;

s3, cell culture: adding culture solution into the centrifugal tube, standing, changing the culture solution in a half-liquid changing mode every 1-3 days until cells grow out from the periphery of the tissue block, changing the culture solution and discarding the tissue block;

s4, cell purification: and (4) removing the fibroblasts in the centrifuge tube in the step S3 by a differential wall pasting method to obtain the purified osteoblasts.

Further, the specific process of the enzymatic digestion of step S2 is as follows:

1) respectively transferring the tissue blocks with the consistent volumes in the step S1 into a centrifugal tube, adding 0.25% trypsin solution with the volume 5-6 times that of the tissue blocks into the centrifugal tube, placing the centrifugal tube into a constant-temperature water bath kettle for oscillating digestion, blowing the tissue blocks to make the digested cells fall off, removing the upper solution after centrifugation, and cleaning with PBS solution;

2) after the cleaning is finished, adding type I collagenase which is 5-6 times of the volume of the tissue block, continuously oscillating and digesting in a constant-temperature water bath kettle, centrifuging and removing an upper layer solution;

3) repeating the step 2) until the tissue blocks are loosened into floccules.

Further, in the step 1), the shaking digestion time is 20-40 min; in the step 2) and the step 3), the time of shaking digestion is 15-25 min.

Further, the temperature of the constant-temperature water bath is 34-37 ℃.

Further, in step S1, the tissue mass has a size of 1-3mm 3.

Further, in step S3, the liquid half-changing method is:

using a rubber-tipped dropper to suck 1ml of the cell culture solution in the centrifuge tube and discard the cell culture solution, and adding 1ml of new culture solution into the centrifuge tube.

Further, in step S4, the differential wall attaching method specifically includes:

sucking old culture solution in a centrifuge tube, discarding, changing the culture solution every 24-36h, digesting with 0.25% trypsin solution, adding equal amount of culture solution to terminate digestion, and blowing and beating cells on the bottle wall and the wall corner to disperse the cells to form cell suspension;

the centrifuge tube was transferred to a six-well plate at 37 ℃ with 5% CO₂After culturing in the incubator, sucking out culture solution and nonadherent cells from the centrifuge tube, centrifuging, adding cell culture solution, suspending, transferring into a new six-well plate, and culturing at 37 deg.C under 5% CO₂Culturing in an incubator to obtain the purified osteoblasts.

The invention has the beneficial effects that:

the culture method used in the invention greatly improves the cell purity of the separated osteoblasts compared with the comparative example, and simultaneously greatly shortens the time required by cell culture.

Drawings

FIG. 1 is a diagram of osteoblasts under 100-fold microscope according to example 1 of the present invention;

FIG. 2 is a diagram of osteoblasts of comparative example 1 under 100-fold microscope;

fig. 3 is a diagram of osteoblasts of comparative example 2 under 100-fold microscope.

Detailed Description

The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.

The invention relates to a method for separating osteoblasts by combining enzyme digestion and tissue block culture,the method comprises the following steps: s1, early preparation: cleaning skull of suckling mouse within seven days in PBS containing double antibody, removing periosteum, cleaning with PBS containing double antibody, and cutting into 1-3mm pieces³The tissue mass of (a); s2, enzyme digestion: (1) respectively transferring the tissue blocks into a centrifugal tube, adding 0.25% trypsin solution which is 5-6 times the volume of the tissue blocks, placing the centrifugal tube into a constant-temperature water bath kettle at the temperature of 34-37 ℃ for shaking digestion for 20-40min, blowing the tissue blocks to make the digested cells fall off, centrifuging for 5min at 2000r/min, removing the upper solution, and cleaning with PBS solution; (2) adding collagenase type I5-6 times the volume of the tissue mass, performing oscillatory digestion in a constant temperature water bath at 34-37 deg.C for 15-25min, centrifuging at 2000r/min for 5min, removing the upper solution, and (3) repeating the above step (2) until the tissue mass is loosened into flocculent; wherein the shaking digestion time in step (1) is preferably more than the shaking digestion time in step (2); s3, cell culture: adding 2mL of culture solution into the centrifugal tube, moving the centrifugal tube into a six-hole plate, standing, and maintaining cell growth every 2 days by using a half-liquid-changing mode, wherein the half-liquid-changing mode is that 1mL of cell culture solution in the centrifugal tube on the six-hole plate is absorbed by using a rubber-head dropper and discarded, adding 1mL of new culture solution into the centrifugal tube until cells grow out from the periphery of a tissue block, and changing the culture solution and discarding the tissue block; s4, cell purification: removing fibroblasts in the centrifugal tube by adopting a differential wall pasting method, wherein the differential wall pasting method comprises the following steps: sucking old culture solution in centrifuge tube, discarding, replacing 1mL every other day, digesting with 0.25% trypsin solution, adding equal amount of culture solution to terminate digestion, blowing and beating cells adhered to bottle wall and wall corner to disperse to form cell suspension, transferring cell suspension into six-well plate, and treating with 5% CO at 37 deg.C to obtain cell suspension₂After culturing in the incubator, sucking out culture solution and nonadherent cells from the centrifuge tube, centrifuging, adding cell culture solution, suspending, transferring into a new six-well plate, and culturing at 37 deg.C under 5% CO₂Culturing in an incubator to obtain osteoblasts, and performing cell passage.

The culture solution used in the above was RPMI1640 culture solution.

Example 1

The culture method comprises the following steps:

s1, early preparation: cleaning skull of suckling mouse within seven days old in PBS containing double antibody, removing periosteum, cleaning with PBS containing double antibody, and cutting into pieces with volume of 3mm³The tissue mass of (a);

s2, enzyme digestion: (1) respectively transferring the tissue blocks into a centrifugal tube, adding 0.25% trypsin solution with the volume 5 times that of the tissue blocks, placing the centrifugal tube into a constant-temperature water bath kettle with the temperature of 37 ℃ for shaking digestion for 20min, blowing the tissue blocks to enable digested cells to fall off, centrifuging for 5min at 2000r/min, removing the upper layer solution, and cleaning by using PBS solution; (2) adding type I collagenase 5 times the volume of the tissue block, digesting for 15min in a constant temperature water bath kettle at 37 ℃, centrifuging for 5min at 2000r/min, removing the upper solution, and (3) repeating the step (2) for 3 times, wherein the tissue block is loosened into flocculent;

s3, cell culture: adding 2mL of RPMI1640 culture solution into a centrifugal tube, moving the centrifugal tube into a six-hole plate, standing, and maintaining cell growth every 2 days by using a half-liquid-changing mode, wherein the half-liquid-changing mode is that 1mL of cell culture solution in the centrifugal tube on the six-hole plate is absorbed by using a rubber head dropper and discarded, then adding 1mL of new culture solution into the centrifugal tube until cells grow out from the periphery of a tissue block, and changing the culture solution and discarding the tissue block;

s4, cell purification: removing fibroblasts in the centrifugal tube by adopting a differential wall pasting method, wherein the differential wall pasting method comprises the following steps: sucking old culture solution in centrifuge tube, discarding, replacing 1mL every other day, digesting with 0.25% trypsin solution, adding equal amount of culture solution to terminate digestion, blowing and beating cells adhered to bottle wall and wall corner to disperse to form cell suspension, transferring cell suspension into six-well plate, and treating with 5% CO at 37 deg.C to obtain cell suspension₂After culturing in the incubator, sucking out culture solution and nonadherent fibroblasts from the centrifuge tube, centrifuging, adding cell culture solution, suspending, transferring into a new six-well plate, and culturing at 37 deg.C and 5% CO₂Culturing in an incubator to obtain cellsBone cells, and can be subjected to cell passaging.

FIG. 1 shows osteoblasts isolated by the above steps, which are anchorage-dependent cells, and have abundant quantity and good growth state, wherein the cell dense region has radial and spiral shapes, and has various irregular shapes, and long spindle shape, and a small amount of dead cells are suspended in the cell fluid; when the cells are close to being fused, the morphology tends to be single, and the short spindle type is mainly used.

Example 2

The culture method comprises the following steps:

s1, early preparation: cleaning skull of suckling mouse within seven days old in PBS containing double antibody, removing periosteum, cleaning with PBS containing double antibody, and cutting into pieces with volume of 3mm³The tissue mass of (a);

s2, enzyme digestion: (1) respectively transferring the tissue blocks into a centrifugal tube, adding 0.25% trypsin solution with the volume 5 times that of the tissue blocks, placing the centrifugal tube into a constant-temperature water bath kettle with the temperature of 37 ℃ for shaking digestion for 20min, blowing the tissue blocks to enable digested cells to fall off, centrifuging for 5min at 2000r/min, removing the upper layer solution, and cleaning by using PBS solution; (2) adding type I collagenase with the volume 5 times that of the tissue blocks, oscillating and digesting in a constant-temperature water bath kettle at 37 ℃ for 25min, centrifuging for 5min at 2000r/min, removing the upper solution, and (3) repeating the step (2) for 3 times, wherein the tissue blocks are loosened into floccules;

step S3 and step S4 are the same as in embodiment 1.

Example 3

The culture method comprises the following steps:

s1, early preparation: cleaning skull of suckling mouse within seven days old in PBS containing double antibody, removing periosteum, cleaning with PBS containing double antibody, and cutting into pieces with volume of 3mm³The tissue mass of (a);

s2, enzyme digestion: (1) respectively transferring the tissue blocks into a centrifugal tube, adding a 0.25% trypsin solution with the volume 6 times that of the tissue blocks, placing the centrifugal tube into a constant-temperature water bath kettle with the temperature of 37 ℃ for shaking digestion for 30min, blowing the tissue blocks to enable digested cells to fall off, centrifuging for 5min at 2000r/min, removing an upper layer solution, and cleaning by using a PBS solution; (2) adding type I collagenase 6 times the volume of the tissue block, performing oscillatory digestion in a constant-temperature water bath kettle at 37 ℃ for 25min, centrifuging at 2000r/min for 5min, removing the upper solution, and (3) repeating the step (2) for 3 times to loosen the tissue block into flocculent;

step S3 and step S4 are the same as in embodiment 1.

Example 4

The culture method comprises the following steps:

s1, early preparation: cleaning skull of suckling mouse within seven days old in PBS containing double antibody, removing periosteum, cleaning with PBS containing double antibody, and cutting into 1mm³The tissue mass of (a);

s2, enzyme digestion: (1) respectively transferring the tissue blocks into a centrifugal tube, adding 0.25% trypsin solution with the volume 5.5 times that of the tissue blocks, placing the centrifugal tube into a constant-temperature water bath kettle with the temperature of 34 ℃ for carrying out oscillatory digestion for 25min, blowing the tissue blocks to enable digested cells to fall off, centrifuging for 5min at 2000r/min, removing the upper layer solution, and cleaning by using PBS solution; (2) adding collagenase type I with volume 5.5 times of the tissue mass, digesting in a constant temperature water bath at 35 deg.C for 20min, centrifuging at 2000r/min for 5min, removing the upper solution, and (3) repeating the step (2) for 3 times to loosen the tissue mass into flocculent;

step S3 and step S4 are the same as in embodiment 1.

Example 5

The culture method comprises the following steps:

s1, early preparation: cleaning skull of suckling mouse within seven days old in PBS containing double antibody, removing periosteum, cleaning with PBS containing double antibody, and cutting into 2mm pieces³The tissue mass of (a);

s2, enzyme digestion: (1) respectively transferring the tissue blocks into a centrifugal tube, adding a 0.25% trypsin solution with the volume 6 times that of the tissue blocks, placing the centrifugal tube into a constant-temperature water bath kettle with the temperature of 34 ℃ for shaking digestion for 40min, blowing the tissue blocks to enable digested cells to fall off, centrifuging for 5min at 2000r/min, removing an upper layer solution, and cleaning by using a PBS solution; (2) adding type I collagenase 5 times the volume of the tissue mass, performing oscillatory digestion in a constant temperature water bath kettle at 34 ℃ for 25min, centrifuging at 2000r/min for 5min, removing the upper layer solution, and (3) repeating the step (2) for 3 times to loosen the tissue mass into flocculent;

step S3 and step S4 are the same as in embodiment 1.

Example 6

The culture method comprises the following steps:

s1, early preparation: cleaning skull of suckling mouse within seven days old in PBS containing double antibody, removing periosteum, cleaning with PBS containing double antibody, and cutting into 2mm pieces³The tissue mass of (a);

s2, enzyme digestion: (1) respectively transferring the tissue blocks into a centrifugal tube, adding a 0.25% trypsin solution with the volume 6 times that of the tissue blocks, placing the centrifugal tube into a constant-temperature water bath kettle with the temperature of 34 ℃ for carrying out oscillatory digestion for 25min, blowing the tissue blocks to enable digested cells to fall off, centrifuging for 5min at 2000r/min, removing an upper layer solution, and cleaning by using a PBS solution; (2) adding type I collagenase 5 times the volume of the tissue mass, performing oscillatory digestion in a constant temperature water bath kettle at 34 ℃ for 25min, centrifuging at 2000r/min for 5min, removing the upper layer solution, and (3) repeating the step (2) for 3 times to loosen the tissue mass into flocculent;

step S3 and step S4 are the same as in embodiment 1.

Comparative example 1

And (3) a tissue block method: placing the skull of a seven-day-old suckling mouse in a sterile culture dish containing double-antibody PBS, removing intrabony tissue and periosteum under a microscope, repeatedly washing with PBS, removing impurities and blood, and cutting into pieces of 1.0-3.0mm³Pieces of size, directly inoculated homogeneously and 25cm²In the culture flask of (1), the inverted culture flask was placed at 37 ℃ with a volume fraction of 5% CO₂Culturing in incubator for about 3h, turning over the culture bottle, adding DMEM culture solution containing fetal calf serum with volume fraction of 15%, continuing culturing, changing the culture solution after 3d, and performing subculture after the cells are fused into a monolayer.

FIG. 2 shows osteoblasts isolated and cultured by tissue block method: the number of osteoblasts that are free from the tissue mass is small and the growth state is poor.

Comparative example 2

An enzyme digestion method: placing the skull of a seven-day-old suckling mouse in a sterile culture dish containing double-antibody PBS, removing intrabony tissue and periosteum under a microscope, repeatedly washing with PBS, removing impurities and blood, and cutting into pieces of 1.0-3.0mm³Size pieces; further adding 0.25% trypsin and containing 0.02% EDTA at 37 deg.C with volume fraction of 5% CO₂Digesting the incubators for 30min respectively, repeatedly digesting for two or three times, continuously shaking a constant temperature shaking table, centrifuging for 5min at 2000r/min, removing trypsin, adding complete culture medium DMEM containing fetal calf serum with volume fraction of 15%, suspending cells at 37 deg.C and 5% CO₂Culturing in an incubator, changing the culture solution after 3d, and carrying out subculture when the cell fusion reaches about 75%.

FIG. 3 shows osteoblasts isolated and cultured by enzymatic digestion: although the number of osteoblasts digested by pure 0.25% pancreatin is large, the pancreatin digestion capability is strong, so that the cells die more and the number of the survival osteoblasts is small.

The isolation culture methods of examples 1 to 6 were compared with those of comparative examples 1 and 2, and the results are shown in Table 1.

TABLE 1

As can be seen from the data in Table 1, the cell purity of the method of the present invention is much improved compared to comparative examples 1 and 2, and the time required for cell culture is also greatly shortened.

While the invention has been described with respect to a preferred embodiment, it will be understood by those skilled in the art that the foregoing and other changes, omissions and deviations in the form and detail thereof may be made without departing from the scope of this invention. Those skilled in the art can make various changes, modifications and equivalent arrangements, which are equivalent to the embodiments of the present invention, without departing from the spirit and scope of the present invention, and which may be made by utilizing the techniques disclosed above; meanwhile, any changes, modifications and variations of the above-described embodiments, which are equivalent to those of the technical spirit of the present invention, are within the scope of the technical solution of the present invention.