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CN108265021B - Leech cell in-vitro culture medium and culture method thereof - Google Patents

Leech cell in-vitro culture medium and culture method thereof Download PDF

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CN108265021B
CN108265021B CN201810268999.6A CN201810268999A CN108265021B CN 108265021 B CN108265021 B CN 108265021B CN 201810268999 A CN201810268999 A CN 201810268999A CN 108265021 B CN108265021 B CN 108265021B
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虞龙
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Changzhou Biotechnology Jiangsu Co ltd
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Abstract

The invention discloses a leech cell in-vitro culture medium and a culture method thereof, wherein a leech tissue block after being cut into pieces is inoculated into a cell closed culture bottle, an Insect-cell culture medium is added, the culture is kept still in a cell culture box, then the separation culture is carried out by matching with a tissue block recovery method, and finally, purified leech somatic cells are obtained by a trypsin digestion method for further culture. Compared with other insect cell culture media, the method can more quickly and successfully obtain leech purified somatic cells, prepare for further carrying out leech cell in-vitro subculture, and build a set of in-vitro primary culture leech cell system.

Description

Leech cell in-vitro culture medium and culture method thereof
Technical Field
The invention relates to a leech in-vitro culture method, and belongs to the field of bioengineering.
Background
With the rapid development of life science, cell culture is an indispensable process for the whole biological engineering technology or one of the biological cloning technologies, and the development of the fields of cell biology, molecular biology, biochemistry and the like is promoted.
Leeches (Whitmania pigra Whitman) belong to the family of leeches of the class Hirudinidae of the phylum Annelida, and according to statistics, more than 600 species of leeches are distributed in the world, and nearly 100 species are distributed in China. Leeches belong to animals in the cold blood link, can grow and breed in south and north China, and mainly live in reservoirs, ditches, paddy fields and lakes and marshes in fresh water, and are most in ponds rich in organic matters or small rivers free of pollution. The optimum growth temperature is 10-40 deg.C, when the temperature is lower than 3 deg.C in northern area, the soil enters into hibernation period, and the hibernation activity is about more than 8 deg.C in 3-4 months of the next year. Leeches are omnivorous animals, which take blood or body fluid of animals as a main life style, plankton, insects and mollusks in water are usually used as main baits, and various animal internal organs, cooked egg yolks, compound feed, plant residues, freshwater snail, trash fish, earthworms and the like are used as baits under artificial conditions.
Researches show that the leech salivary gland secretion contains various bioactive substances such as hirudin and the like and has wide application prospect in medicine. Hirudin, leech hyaluronidase, antipixin, anesthetic, vasodilator, prostaglandin and the like, has the effects of reducing blood fat, protecting heart and cerebral vessels, resisting blood coagulation, resisting tumors and the like, and has important research significance. Therefore, there is a great clinical need for natural hirudin. The existing hirudin extraction mainly adopts biological engineering means to recombine and stimulate leech salivary gland to secrete hirudin, and the two methods have high cost and extremely low yield of the obtained hirudin. With the development of cell in vitro culture technology, obtaining hirudin by culturing leech cells in vitro becomes a popular research subject, and researches show that the hirudin obtained by the method not only has high yield, but also has the effect which is comparable to the hirudin obtained by stimulating salivary gland secretion of leech. However, the current leech cell in-vitro culture technology is still in the sprouting stage, and the selection of a culture medium, the determination of culture conditions, the selection of antibiotics and the like are all difficult problems which need to be overcome urgently.
Disclosure of Invention
Aiming at the problems, the invention provides a leech cell in-vitro culture method.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
the leech cell in-vitro culture medium is characterized by comprising the following components in parts by weight:
inorganic salts:
NaCl1420mg/L，ZnCL2 876.54mg/L，MgSO4·7H2O1743.48mg/L，KCl2080mg/L，KH2PO4·2H2O 1000mg/L；
amino acids:
9000mg/L of DL-serine, 620mg/L of glycine, 700mg/L of L-arginine hydrochloride, 400mg/L of L-asparagine, 350mg/L of L-aspartic acid, 160mg/L of L-cysteine dihydrochloride, 3500mg/L of L-glutamic acid, 2000mg/L of L-histidine hydrochloride, 200mg/L of L-isoleucine, 150mg/L of L-leucine, 850mg/L of L-lysine hydrochloride, 100mg/L of L-methionine, 100mg/L of L-phenylalanine, 500mg/L of L-proline, 200mg/L of L-threonine, 150mg/L of L-tryptophan, 580mg/L of disodium hydrogen phosphate L-tyrosine, l-valine 200mg/L, beta-alanine 400mg/L and L-alanine 225 mg/L;
vitamins:
0.15mg/L of D-biotin, 0.15mg/L of D-calcium pantothenate, 0.15mg/L of folate, 0.10mg/L of cyanocobalamine, 0.15mg/L of nicotinic acid, 0.15mg/L of pyridoxamine hydrochloride, 0.15mg/L of riboflavin, 0.10mg/L of thiamine and 0.15mg/L of p-aminobenzoic acid;
and (3) the other:
2000mg/L of sucrose, 1500mg/L of D-glucose, 500mg/L of D-fructose, 600mg/L of maltose, 330mg/L of alpha-ketoglutaric acid, 90mg/L of fumaric acid, 90mg/L of succinic acid and 90mg/L of L-malic acid; the pH was adjusted to 7.4 with 1.0N KOH.
A leech cell in vitro culture method comprises inoculating minced leech tissue blocks into a cell closed culture bottle, adding the Insect-cell culture medium, performing static culture in a cell culture box, performing separation culture by using a tissue block recovery method, and performing further culture on purified leech somatic cells obtained by a trypsin digestion method. The method comprises the following specific steps
1) Soaking living Hirudo in normal saline for 1 hr to remove impurities and ensure somatic cell activity to the maximum extent, cleaning surface mucosa with normal saline, washing with distilled water for three times, soaking in 70% ethanol for 5min to remove bacteria on body surface, and washing with distilled water repeatedly to remove ethanol;
2) transferring the leech subjected to the epidermis sterilization treatment in the step 1) into an ultraclean workbench, putting the leech into a sterile culture dish containing sterile PBS, and shearing two ends of a leech body by using sterile scissors and tweezers to take head tissues;
3) transferring the head tissue in the step 2) into another sterile culture dish filled with PBS buffer solution, and soaking the head tissue in the leech body; dissecting the head tissue with a sterile scalpel, taking the tissue in the middle of the epidermis, and washing with a sterile PBS buffer solution;
4) soaking the tissue in the step 3) in 75% alcohol for 1min, taking out, rinsing with sterile PBS buffer solution, and repeating for three times;
5) placing the tissue in the step 4) in a sterile culture dish containing sterile PBS, shearing the tissue into small tissue blocks by using sterile scissors and tweezers, and soaking for 5 min;
6) removing the tissue debris floating on the upper part of the fine tissue block in the step 5), sucking the tissue block paste by using a 5mL gun head, paving the sucked paste tissue at the bottom of a bottle, uniformly distributing the tissue block paste in a 25mL cell closed culture bottle, adding 5mL of an Insect-CCulture Insect cell culture medium, and standing and culturing for 60min in a 25 ℃ cell culture box for adherence.
7) Sucking the excess liquid at the bottom of the culture bottle after the static adherent culture for 60min in the step 6) by using a suction pipe, slowly adding 5mL of an Insect-cell culture medium into the bottle mouth by using a liquid-transferring gun, covering a tissue block, continuing primary culture, and timely replacing fresh culture solution when the color of the culture solution becomes dark; after primary culture for 6-12h, when the bottom of the bottle is about to be filled with cells, removing the tissue blocks, re-inoculating the removed tissue blocks into a new culture bottle, culturing in an incubator, and timely replacing fresh culture solution.
8) Discarding the culture solution of the cells obtained in the step 7), cleaning the cells by sterile PBS, adding 2mL of pancreatin digestive juice, digesting the cells for 50s, slightly shaking the culture bottle, observing the cells under a microscope, immediately adding 5mL of an institute-CCulture Insect cell culture medium to stop digestion when the fibroblasts shrink to become round and float and the leech somatic cells shrink but do not float, slightly shaking the cells to discard the culture solution, adding 4mL of PBS, slightly washing the residual fibroblasts, adding 2mL of digestive juice, digesting the cells for 3min, discarding 1mL of the digestive juice, beating the bottom of the bottle from the bottom to completely drop the cells, adding 5mL of a Lanselect-CCulture Insect cell culture medium to stop digestion, repeatedly blowing the cells to disperse cell clusters into single cells.
9) And (3) filtering the cells obtained from the cells obtained in the step (8) by using eight layers of gauze, and inoculating the filtered cells into a 25mL culture bottle containing 5mL of Insect-cell culture medium with the culture temperature of 25 ℃ for 3 days.
The culture medium of the Insect cell of the culture medium Insect-CCulture used by the invention has the following formula:
inorganic salts:
NaCl1420mg/L，ZnCL2 876.54mg/L，MgSO4·7H2O1743.48mg/L，KCl2080mg/L，KH2PO4·2H2O 1000mg/L；
amino acids:
9000mg/L of DL-serine, 620mg/L of glycine, 700mg/L of L-arginine hydrochloride, 400mg/L of L-asparagine, 350mg/L of L-aspartic acid, 160mg/L of L-cysteine dihydrochloride, 3500mg/L of L-glutamic acid, 2000mg/L of L-histidine hydrochloride, 200mg/L of L-isoleucine, 150mg/L of L-leucine, 850mg/L of L-lysine hydrochloride, 100mg/L of L-methionine, 100mg/L of L-phenylalanine, 500mg/L of L-proline, 200mg/L of L-threonine, 150mg/L of L-tryptophan, 580mg/L of disodium hydrogen phosphate L-tyrosine, l-valine 200mg/L, beta-alanine 400mg/L and L-alanine 225 mg/L;
vitamins:
0.15mg/L of D-biotin, 0.15mg/L of D-calcium pantothenate, 0.15mg/L of folate, 0.10mg/L of cyanocobalamine, 0.15mg/L of nicotinic acid, 0.15mg/L of pyridoxamine hydrochloride, 0.15mg/L of riboflavin, 0.10mg/L of thiamine and 0.15mg/L of p-aminobenzoic acid;
and (3) the other:
2000mg/L of sucrose, 1500mg/L of D-glucose, 500mg/L of D-fructose, 600mg/L of maltose, 330mg/L of alpha-ketoglutaric acid, 90mg/L of fumaric acid, 90mg/L of succinic acid and 90mg/L of L-malic acid;
the pH was adjusted to 7.4 with 1.0N KOH and 15% inactivated insect-specific special grade fetal calf serum was added before use.
The content of penicillin added into the sterile PBS buffer solution used in the invention is 600U/mL, the content of streptomycin is 600U/mL, and the content of amphotericin B is 15 mug/mL; the size of the tissue block after being cut into pieces is 1mm3。
The antibiotic-containing Insect cell culture medium used in the invention is additionally added with special grade fetal calf serum with penicillin content of 600U/mL, streptomycin content of 600U/mL, amphotericin B content of 15 mu g/mL and inactivated 15%.
The invention has the beneficial effects that: the invention provides a cell in-vitro culture method for primary culture of leech cells, and also provides an optimized insect cell culture medium, which is used for obtaining leech purified somatic cells more quickly and successfully compared with other insect cell culture media, preparing for further carrying out in-vitro subculture of leech cells and constructing a set of in-vitro primary culture leech cell system. The invention provides accurate and reliable experimental basis for the in vitro culture of the leech cells.
Detailed Description
The invention will be better understood from the following examples. However, those skilled in the art will readily appreciate that the specific material ratios, process conditions and results described in the examples are merely illustrative of the invention and should not be construed as limiting the invention.
The leeches used in the following examples were derived from: hirudo nipponica.
Example 1
The invention provides a leech living body growth cycle selection method which comprises the following specific steps
The method comprises the following steps: soaking 1, 2 and 3 years old living leeches in normal saline for 1h respectively, primarily removing impurities in vivo and ensuring the vitality of somatic cells to the maximum extent, cleaning surface mucosa with normal saline until the surface mucosa is clean, finally cleaning with distilled water for three times, soaking in 70% alcohol for 5min, removing body surface bacteria, and repeatedly washing with distilled water to remove alcohol;
step two: transferring the leeches respectively subjected to the epidermis sterilization treatment in the step one into an ultraclean workbench, putting the leeches into a sterile culture dish containing sterile PBS, and shearing two ends of leeches by using sterile scissors and tweezers to take head tissues;
step three: transferring the head tissue in the second step into another sterile culture dish filled with PBS buffer solution, and soaking the leech body;
step four: respectively dissecting the head tissues in the third step by using a sterile scalpel, taking the tissues in the middle of the epidermis, and washing by using a sterile PBS buffer solution;
step five: soaking the tissue in the fourth step in 75% alcohol for 1min, taking out, rinsing with sterile PBS buffer solution, and repeating for three times;
step six: placing the tissue in the fifth step into a sterile culture dish containing sterile PBS, shearing the tissue into small tissue blocks by using sterile scissors and tweezers, and soaking for 5 min;
step seven: removing the tissue debris floating on the upper part of the fine tissue block in the sixth step, respectively sucking the tissue block paste by using a 5mL gun head, paving the sucked paste tissue at the bottom of a bottle, uniformly distributing the tissue block paste in a 25mL cell closed culture bottle, adding 5mL Insect-cell culture medium containing antibiotics into the bottle, and standing and culturing for 60min in a 25 ℃ cell culture box.
Step eight: absorbing excess liquid at the bottom of the culture bottle after the culture bottle is subjected to static adherent culture for 60min by using a suction tube, slowly adding 5mL of Insect-cell culture medium containing antibiotics into the bottle mouth by using a liquid-transferring gun, covering a tissue block, and timely replacing fresh culture solution when the color of the culture solution becomes dark; after primary culture for 6-12h, when the bottom of the bottle is about to be filled with cells, removing the tissue blocks, re-inoculating the removed tissue blocks into a new culture bottle, culturing in an incubator, and timely replacing fresh culture solution.
Step nine: respectively removing culture solution from the cells obtained in the step eight, cleaning the cells by using sterile PBS, adding 2mL of pancreatin digestive juice, digesting for 50s, slightly shaking the culture bottle, observing under a microscope, immediately adding 5mL of an antibiotic-containing Insect cell culture medium to stop digestion when the leech somatic cells shrink but do not float, slightly shaking the culture bottle to remove the culture solution, adding 4mL of PBS, slightly washing residual fibroblasts, adding 2mL of digestive juice, digesting for 3min, removing 1mL of digestive juice, beating the bottom of the bottle from the bottom to completely drop the cells, adding 6mL of the antibiotic-containing Insect cell culture medium to stop digestion, and repeatedly blowing the cells to disperse cell clusters into single cells.
Step ten: and (4) filtering the cells obtained from the cell obtained in the ninth step by using eight layers of gauze, inoculating the filtered cells into a 25mL culture bottle containing 5mL of the Insect cell culture medium of the Insect-shaped cell, culturing at the temperature of 25 ℃ for 3 days, continuing transferring the cells into another 25mL culture bottle containing 5mL of the Insect-shaped cell culture medium of the Insect-shaped cell, and repeating the previous operation.
The culture medium of the Insect cell of the culture medium Insect-CCulture used by the invention has the following formula:
inorganic salts:
NaCl1420mg/L，ZnCL2 876.54mg/L，MgSO4·7H2O1743.48mg/L，
KCl2080mg/L，KH2PO4·2H2O 1000mg/L；
amino acids:
9000mg/L of DL-serine, 620mg/L of glycine, 700mg/L of L-arginine hydrochloride, 400mg/L of L-asparagine, 350mg/L of L-aspartic acid, 160mg/L of L-cysteine dihydrochloride, 3500mg/L of L-glutamic acid, 2000mg/L of L-histidine hydrochloride, 200mg/L of L-isoleucine, 150mg/L of L-leucine, 850mg/L of L-lysine hydrochloride, 100mg/L of L-methionine, 100mg/L of L-phenylalanine, 500mg/L of L-proline, 200mg/L of L-threonine, 150mg/L of L-tryptophan, 580mg/L of disodium hydrogen phosphate L-tyrosine, l-valine 200mg/L, beta-alanine 400mg/L and L-alanine 225 mg/L;
vitamins:
0.15mg/L of D-biotin, 0.15mg/L of D-calcium pantothenate, 0.15mg/L of folate, 0.10mg/L of cyanocobalamine, 0.15mg/L of nicotinic acid, 0.15mg/L of pyridoxamine hydrochloride, 0.15mg/L of riboflavin, 0.10mg/L of thiamine and 0.15mg/L of p-aminobenzoic acid;
and (3) the other:
2000mg/L of sucrose, 1500mg/L of D-glucose, 500mg/L of D-fructose, 600mg/L of maltose, 330mg/L of alpha-ketoglutaric acid, 90mg/L of fumaric acid, 90mg/L of succinic acid and 90mg/L of L-malic acid;
the pH was adjusted to 7.4 with 1.0N KOH and 15% inactivated insect-specific special grade fetal calf serum was added before use.
The content of penicillin added into the sterile PBS buffer solution used in the invention is 600U/mL, the content of streptomycin is 600U/mL, and the content of amphotericin B is 15 mug/mL; the size of the tissue block after being cut into pieces is 1mm3。
The antibiotic-containing Insect cell culture medium used in the invention is additionally added with special grade fetal calf serum with penicillin content of 600U/mL, streptomycin content of 600U/mL, amphotericin B content of 15 mu g/mL and inactivated 15%.
This example shows that selection of 2 year old samples resulted in free cells after 10h of primary culture and subsequent adherent growth of subcultured cells, and that 1, 3 year old samples showed no free cells after 15h of primary culture. The invention can be used as an accurate basis for selecting leech living bodies.
Example 2
The invention provides an insect cell culture medium optimized for leech cell primary culture, which comprises the following specific steps
The method comprises the following steps: soaking living Hirudo in normal saline for 1 hr to remove impurities and ensure somatic cell activity to the maximum extent, cleaning surface mucosa with normal saline, washing with distilled water for three times, soaking in 70% ethanol for 5min to remove bacteria on body surface, and washing with distilled water repeatedly to remove ethanol;
step two: transferring the leech subjected to the epidermis sterilization treatment in the step one into an ultraclean workbench, putting the leech into a sterile culture dish containing 600U/mL of PBS containing different antibiotics, and shearing two ends of a leech body by using sterile scissors and tweezers to take head tissues;
step three: transferring the head tissue in the second step into another sterile culture dish filled with PBS buffer solution, and soaking the leech body;
step four: dissecting the head tissue in the third step with a sterile scalpel, taking the tissue in the middle of the epidermis, and washing with a sterile PBS buffer solution;
step five: soaking the tissue in the fourth step in 75% alcohol for 1min, taking out, rinsing with PBS buffer solution containing 600U/mL different antibiotics, and repeating for three times;
step six: placing the tissue in the fifth step into a sterile culture dish containing 600U/mL PBS containing different antibiotics, shearing the tissue into small tissue blocks by using sterile scissors and tweezers, and soaking for 5 min;
step seven: removing the tissue debris floating on the upper part of the fine tissue block in the sixth step, sucking the tissue block paste by using a 5mL gun head, paving the sucked paste tissue at the bottom of a bottle, uniformly distributing the tissue block paste in a 25mL cell closed culture bottle, adding 5mL of an Insect-CCulture Insect cell culture medium containing 600U/mL of different antibiotics, and carrying out standing culture in a 25 ℃ cell culture box for 60 min.
Step eight: absorbing excess liquid at the bottom of the culture bottle after the culture bottle is subjected to static adherent culture for 60min by using a suction pipe, slowly adding 5mL of Insect-cell culture medium containing 600U/mL of different antibiotics, SFX-Instrument and MGM-450 into the bottle mouth by using a liquid-transferring gun, covering tissue blocks, and timely replacing fresh culture solution when the color of the culture solution becomes dark; after culturing for 6-12h, when the bottom of the bottle is about to be filled with cells, removing the tissue blocks, re-inoculating the removed tissue blocks into a new culture bottle, culturing in an incubator, timely replacing fresh culture solution, and observing the growth condition of the cells.
Step nine: removing the culture solution from the cells obtained in the step eight, washing the cells by sterile PBS, adding 2mL of pancreatin digestive juice, digesting the cells for 50s, slightly shaking the culture bottle, observing the cells under a microscope until the leech somatic cells shrink but do not float, immediately adding 5mL of Insect cell culture medium containing 600U/mL of different antibiotics for treating infection-CCulture, SFX-institute and MGM-450, respectively, stopping digestion, slightly shaking, discarding the culture solution, adding 4mL of PBS, slightly washing to remove residual fibroblasts, adding 2mL of digestive juice, digesting for 3min, discarding 1mL of digestive juice, beating the bottom of the flask from the bottom to completely drop the cells, adding 5mL of Insect-cell culture medium containing 600U/mL of different antibiotics, SFX-Instrument and MGM-450 respectively to stop digestion, and repeatedly blowing and beating the cells to disperse cell clusters into single cells.
Step ten: and (4) filtering the cells obtained from the cell obtained in the ninth step by using eight layers of gauze, inoculating the filtered cells into a 25mL culture bottle containing 5mL of the Insect cell culture medium of the Insect-shaped cell, culturing at the temperature of 25 ℃ for 3 days, continuing transferring the cells into another 25mL culture bottle containing 5mL of the Insect-shaped cell culture medium of the Insect-shaped cell, and repeating the previous operation.
The culture medium of the Insect cell of the culture medium Insect-CCulture used by the invention has the following formula:
inorganic salts:
NaCl1420mg/L，ZnCL2 876.54mg/L，MgSO4·7H2O1743.48mg/L，
KCl2080mg/L，KH2PO4·2H2O 1000mg/L；
amino acids:
9000mg/L of DL-serine, 620mg/L of glycine, 700mg/L of L-arginine hydrochloride, 400mg/L of L-asparagine, 350mg/L of L-aspartic acid, 160mg/L of L-cysteine dihydrochloride, 3500mg/L of L-glutamic acid, 2000mg/L of L-histidine hydrochloride, 200mg/L of L-isoleucine, 150mg/L of L-leucine, 850mg/L of L-lysine hydrochloride, 100mg/L of L-methionine, 100mg/L of L-phenylalanine, 500mg/L of L-proline, 200mg/L of L-threonine, 150mg/L of L-tryptophan, 580mg/L of disodium hydrogen phosphate L-tyrosine, l-valine 200mg/L, beta-alanine 400mg/L and L-alanine 225 mg/L;
vitamins:
0.15mg/L of D-biotin, 0.15mg/L of D-calcium pantothenate, 0.15mg/L of folate, 0.10mg/L of cyanocobalamine, 0.15mg/L of nicotinic acid, 0.15mg/L of pyridoxamine hydrochloride, 0.15mg/L of riboflavin, 0.10mg/L of thiamine and 0.15mg/L of p-aminobenzoic acid;
and (3) the other:
2000mg/L of sucrose, 1500mg/L of D-glucose, 500mg/L of D-fructose, 600mg/L of maltose, 330mg/L of alpha-ketoglutaric acid, 90mg/L of fumaric acid, 90mg/L of succinic acid and 90mg/L of L-malic acid;
the pH was adjusted to 7.4 with 1.0N KOH and 15% inactivated insect-specific special grade fetal calf serum was added before use.
This example shows that in the 11 th hour of the primary culture of the Insect cell culture medium of the institute-CCulture containing 600U/mL three-antibody mixed solution, a small amount of cells are liberated on the periphery of the tissue block, and the shape is circular and transparent; the culture medium has no pollution, the survival time of the cells is long, and the cells grow adherent to the walls in the subsequent subculture experiment; after the SFX-Insect cell culture medium is subjected to primary culture for 15h, observing that a culture solution is still clear under a microscope, and the cells have no growth signs temporarily; after 15h of primary culture of MGM-450 insect cell culture medium, the culture solution is observed to be light yellow and black spots appear in the culture solution under a microscope, no free cells appear around the tissues, and the cells show temporary growth. Therefore, the optimized and improved Insect cell culture medium of the invention can be used as a culture medium for culturing leech cells.
Example 3
The antibiotic type selection provided by the invention comprises the following specific steps
The method comprises the following steps: soaking living Hirudo in normal saline for 1 hr to remove impurities and ensure somatic cell activity to the maximum extent, cleaning surface mucosa with normal saline, washing with distilled water for three times, soaking in 70% ethanol for 5min to remove bacteria on body surface, and washing with distilled water repeatedly to remove ethanol;
step two: transferring the leech subjected to the epidermis sterilization treatment in the step one into an ultraclean workbench, putting the leech into a sterile culture dish containing different antibiotics (penicillin, streptomycin and amphotericin B) PBS, and shearing two ends of a leech body by using sterile scissors and tweezers to take head tissues;
step three: transferring the head tissue in the second step to another sterile culture dish filled with PBS buffer solution of different antibiotics (penicillin, streptomycin and amphotericin B) and soaking the head tissue in the leech body;
step four: dissecting the head tissue in the third step with a sterile scalpel, taking the tissue in the middle of the epidermis, and washing with PBS (phosphate buffer solution) containing different antibiotics (penicillin, streptomycin and amphotericin B);
step five: soaking the tissue in the fourth step in 75% alcohol for 1min, taking out, rinsing with three-antibody mixed solution (penicillin, streptomycin and amphotericin B) PBS buffer solution with different concentrations, and repeating for three times;
step six: placing the tissue in the fifth step into a sterile culture dish containing different antibiotics (penicillin, streptomycin and amphotericin B) PBS, shearing into small tissue blocks by using sterile scissors and forceps, and soaking for 5 min;
step seven: removing the tissue debris floating on the upper part of the fine tissue block in the sixth step, sucking the tissue block paste by using a 5mL gun head, paving the sucked paste tissue at the bottom of a bottle, uniformly distributing the tissue block paste in a 25mL cell closed culture bottle, adding 5mL of an Insect cell culture medium containing different types of antibiotics (penicillin, streptomycin and amphotericin B) and carrying out standing culture in a 25 ℃ cell culture box for 60 min.
Step eight: absorbing excess liquid at the bottom of the culture bottle after the culture for 60min by using a suction pipe, slowly adding 5mL of an Insect cell culture medium containing different types of antibiotics (penicillin, streptomycin and amphotericin B) into the bottle mouth by using a liquid-transferring gun, covering a tissue block, and timely replacing fresh culture solution when the color of the culture solution becomes dark; after primary culture for 6-12h, when the bottom of the bottle is about to be filled with cells, removing the tissue blocks, re-inoculating the removed tissue blocks into a new culture bottle, culturing in an incubator, and timely replacing fresh culture solution.
Step nine: discarding the culture solution of the cells obtained in the step eight, washing the cells with PBS containing different antibiotics (penicillin, streptomycin and amphotericin B), adding 2mL of pancreatin digestive juice, digesting the cells for 50s, slightly shaking the culture flask, observing under a microscope, immediately adding 5mL of an Insect cell culture medium containing different antibiotics (penicillin, streptomycin and amphotericin B) to terminate digestion when the leech somatic cells shrink but do not float, slightly shaking the cells to discard the culture solution, adding 4mL of PBS containing different antibiotics (penicillin, streptomycin and amphotericin B), slightly washing residual fibroblasts, adding 2mL of digestive juice, digesting the cells for 2-3.5min, discarding 1mL of digestive juice, beating the bottom of the flask to completely drop the cells, adding 5mL of an Insect cell culture medium containing different antibiotics (penicillin, streptomycin and amphotericin B) to terminate digestion, repeatedly blowing and beating the cells to disperse the cell clusters into single cells.
Step ten: and (4) filtering the cells obtained from the ninth step by using eight layers of gauze, inoculating the filtered cells into a 25mL culture bottle containing 5mL of the Insect cell culture medium of the Insect-shaped culture medium of the Insect with different types of antibiotics (penicillin, streptomycin and amphotericin B), culturing at the temperature of 25 ℃ for 3 days, continuing transferring the cells into another 25mL culture bottle containing 5mL of the Insect-shaped culture medium, and repeating the previous operation.
The culture medium of the Insect cell of the culture medium Insect-CCulture used by the invention has the following formula:
inorganic salts:
NaCl1420mg/L，ZnCL2 876.54mg/L，MgSO4·7H2O1743.48mg/L，
KCl2080mg/L，KH2PO4·2H2O 1000mg/L；
amino acids:
9000mg/L of DL-serine, 620mg/L of glycine, 700mg/L of L-arginine hydrochloride, 400mg/L of L-asparagine, 350mg/L of L-aspartic acid, 160mg/L of L-cysteine dihydrochloride, 3500mg/L of L-glutamic acid, 2000mg/L of L-histidine hydrochloride, 200mg/L of L-isoleucine, 150mg/L of L-leucine, 850mg/L of L-lysine hydrochloride, 100mg/L of L-methionine, 100mg/L of L-phenylalanine, 500mg/L of L-proline, 200mg/L of L-threonine, 150mg/L of L-tryptophan, 580mg/L of disodium hydrogen phosphate L-tyrosine, l-valine 200mg/L, beta-alanine 400mg/L and L-alanine 225 mg/L;
vitamins:
0.15mg/L of D-biotin, 0.15mg/L of D-calcium pantothenate, 0.15mg/L of folate, 0.10mg/L of cyanocobalamine, 0.15mg/L of nicotinic acid, 0.15mg/L of pyridoxamine hydrochloride, 0.15mg/L of riboflavin, 0.10mg/L of thiamine and 0.15mg/L of p-aminobenzoic acid;
and (3) the other:
2000mg/L of sucrose, 1500mg/L of D-glucose, 500mg/L of D-fructose, 600mg/L of maltose, 330mg/L of alpha-ketoglutaric acid, 90mg/L of fumaric acid, 90mg/L of succinic acid and 90mg/L of L-malic acid;
the pH was adjusted to 7.4 with 1.0N KOH and 15% inactivated insect-specific special grade fetal calf serum was added before use.
The example shows that when the concentration of the antibiotic in the same type of culture medium is 600U/mL, only under the condition that penicillin, streptomycin and amphotericin B exist at the same time, cells around the tissue grow and free cells appear during primary culture, the culture solution is pollution-free, and a large amount of cells adhere to the wall in subculture, so that the growth condition is good; contamination occurs with penicillin, streptomycin and amphotericin B alone or in combination.
Example 4 selection of antibiotic concentrations provided by the invention comprises the following specific steps
The method comprises the following steps: soaking living Hirudo in normal saline for 1 hr to remove impurities and ensure somatic cell activity to the maximum extent, cleaning surface mucosa with normal saline, washing with distilled water for three times, soaking in 70% ethanol for 5min to remove bacteria on body surface, and washing with distilled water repeatedly to remove ethanol;
step two: transferring the leech subjected to the epidermis sterilization treatment in the step one into an ultraclean workbench, putting the leech into a sterile culture dish containing PBS (penicillin, streptomycin and amphotericin B) mixed liquor with different concentrations, and shearing off two ends of a leech body by using sterile scissors and tweezers to take head tissues;
step three: transferring the head tissue in the second step to another sterile culture dish filled with PBS buffer solution of three antibodies (penicillin, streptomycin and amphotericin B) with different concentrations, and soaking the head tissue in the leech body;
step four: dissecting the head tissue in the third step with a sterile scalpel, taking the tissue in the middle of the epidermis, and washing with PBS (phosphate buffer solution) of three-antibody mixed liquor (penicillin, streptomycin and amphotericin B) with different concentrations;
step five: soaking the tissue in the fourth step in 75% alcohol for 1min, taking out, rinsing with three-antibody mixed solution (penicillin, streptomycin and amphotericin B) PBS buffer solution with different concentrations, and repeating for three times;
step six: placing the tissue in the fifth step into a sterile culture dish containing PBS (mixed solution of three antibiotics with different concentrations) (penicillin, streptomycin and amphotericin B), shearing into small tissue blocks by using sterile scissors and tweezers, and soaking for 5 min;
step seven: removing the tissue debris floating on the upper part of the fine tissue block in the sixth step, sucking the tissue block paste by using a 5mL gun head, paving the sucked paste tissue at the bottom of a bottle, uniformly distributing the tissue block paste in a 25mL cell closed culture bottle, adding 5mL of three-antibody mixed solution (penicillin, streptomycin and amphotericin B) with different concentrations into an Insect cell culture medium with different concentrations, and carrying out standing culture in a 25 ℃ cell culture box for 60 min.
Step eight: absorbing excess liquid at the bottom of the culture bottle after the culture for 60min by using a suction pipe, slowly adding 5mL of an Insect cell culture medium containing three-antibody mixed liquid (penicillin, streptomycin and amphotericin B) with different concentrations into the bottle mouth by using a liquid-transferring gun, covering a tissue block, and timely replacing fresh culture solution when the color of the culture solution becomes dark; after primary culture for 6-12h, when the bottom of the bottle is about to be filled with cells, removing the tissue blocks, re-inoculating the removed tissue blocks into a new culture bottle, culturing in an incubator, and timely replacing fresh culture solution.
Step nine: removing the culture solution from the cells obtained in the step eight, washing by PBS (mixed solution of three antibodies with different concentrations (penicillin, streptomycin and amphotericin B)), adding 2mL of pancreatin digestive juice, digesting for 50s, slightly shaking the culture flask, observing under a microscope, immediately adding 5mL of the input-CCulture Insect cell culture medium containing the mixed solution of three antibodies with different concentrations (penicillin, streptomycin and amphotericin B) to stop digestion when the leech somatic cells shrink but do not float, slightly shaking to remove the culture solution, adding 4mL of PBS (mixed solution of three antibodies with different concentrations (penicillin, streptomycin and amphotericin B) to slightly wash away residual fibroblasts, adding 2mL of digestive juice, digesting for 2-3.5min, removing 1mL of digestive juice, completely beating the cells from the bottom of the bottom flask, adding 5mL of the input-CCulture Insect cell culture medium containing the mixed solution of three antibodies with different concentrations (penicillin, streptomycin and amphotericin B) to stop digestion of the Insect cells, repeatedly blowing and beating the cells to disperse the cell clusters into single cells.
Step ten: and (4) filtering the cells obtained from the ninth step by using eight layers of gauze, inoculating the filtered cells into 25mL culture bottles containing 5mL of Insect-cell culture medium of the infection-CCulture with the three-antibody mixed solution (penicillin, streptomycin and amphotericin B) with different concentrations, culturing at the temperature of 25 ℃ for 3 days, continuing transferring the cells into another 25mL culture bottle containing 5mL of the Insect-cell culture medium of the infection-CCulture, and repeating the previous operation.
The culture medium of the Insect cell of the culture medium Insect-CCulture used by the invention has the following formula:
inorganic salts:
NaCl1420mg/L，ZnCL2 876.54mg/L，MgSO4·7H2O1743.48mg/L，
KCl2080mg/L，KH2PO4·2H2O 1000mg/L；
amino acids:
9000mg/L of DL-serine, 620mg/L of glycine, 700mg/L of L-arginine hydrochloride, 400mg/L of L-asparagine, 350mg/L of L-aspartic acid, 160mg/L of L-cysteine dihydrochloride, 3500mg/L of L-glutamic acid, 2000mg/L of L-histidine hydrochloride, 200mg/L of L-isoleucine, 150mg/L of L-leucine, 850mg/L of L-lysine hydrochloride, 100mg/L of L-methionine, 100mg/L of L-phenylalanine, 500mg/L of L-proline, 200mg/L of L-threonine, 150mg/L of L-tryptophan, 580mg/L of disodium hydrogen phosphate L-tyrosine, l-valine 200mg/L, beta-alanine 400mg/L and L-alanine 225 mg/L;
vitamins:
0.15mg/L of D-biotin, 0.15mg/L of D-calcium pantothenate, 0.15mg/L of folate, 0.10mg/L of cyanocobalamine, 0.15mg/L of nicotinic acid, 0.15mg/L of pyridoxamine hydrochloride, 0.15mg/L of riboflavin, 0.10mg/L of thiamine and 0.15mg/L of p-aminobenzoic acid;
and (3) the other:
2000mg/L of sucrose, 1500mg/L of D-glucose, 500mg/L of D-fructose, 600mg/L of maltose, 330mg/L of alpha-ketoglutaric acid, 90mg/L of fumaric acid, 90mg/L of succinic acid and 90mg/L of L-malic acid;
the pH was adjusted to 7.4 with 1.0N KOH and 15% inactivated insect-specific special grade fetal calf serum was added before use.
This example shows that in the same type of medium, when the penicillin content is 600U/mL, the streptomycin content is 600U/mL, and the amphotericin B content is 15. mu.g/mL, the cell growth condition is the best, and no contamination phenomenon occurs while the cell survival time is long; when the content of penicillin is 200U/mL, the content of streptomycin is 200U/mL and the content of amphotericin B is 5 mug/mL, the turbid phenomenon appears in the culture solution after primary culture for 10 hours, the cells around the tissues are free, and the cells do not grow; when the content of penicillin is 400U/mL, the content of streptomycin is 400U/mL and the content of amphotericin B is 5 mug/mL, the culture solution is light yellow after primary culture for 15 hours, and black granular substances are arranged around tissues, so that no cells are dissociated, and the cells do not grow; when the content of penicillin is 800U/mL, the content of streptomycin is 800U/mL and the content of amphotericin B is 20 mug/mL, the culture solution is clarified after primary culture for 15 hours, no cell is dissociated around the tissue, and the cell does not grow; after 20h of culture, the culture solution is clear, no cells are free around the tissues, and the cells do not grow.

Claims (1)

1.一种水蛭细胞体外培养方法，其特征在于，包括如下步骤：1. a leech cell in vitro culture method, is characterized in that, comprises the steps: 1）将剪碎后的水蛭唾液腺组织块接种于细胞封闭培养瓶中，加入Insect-CCulture 昆虫细胞培养基，25℃细胞培养箱中静置培养60min；1) Inoculate the shredded leech salivary gland tissue blocks into a cell-sealed culture flask, add Insect-CCulture insect cell culture medium, and incubate for 60 minutes in a 25°C cell incubator; 2）配合组织块回收法进行分离培养；2) Separation and culture with tissue block recovery method; 3）通过胰酶消化获得纯化的水蛭体细胞；3) Purified leech somatic cells were obtained by trypsin digestion; 步骤1）中水蛭唾液腺组织块通过如下处理方法获得：Step 1) The leech salivary gland tissue block is obtained by the following processing methods: a、取水蛭活体在生理盐水中浸泡1h，初步去除体内杂质并最大限度保证其体细胞活力，再用生理盐水清洗其表面粘膜至干净，最后用蒸馏水清洗三遍后置于70%的酒精中浸泡5min，去除体表细菌，用蒸馏水重复多次冲洗去酒精；a. Take the leech live and soak it in physiological saline for 1 hour to initially remove impurities in the body and ensure the vitality of its somatic cells to the greatest extent. Then wash the surface mucosa with physiological saline until it is clean, and finally wash it with distilled water for three times and then place it in 70% alcohol Soak for 5 minutes to remove bacteria on the body surface, and repeatedly rinse with distilled water to remove alcohol; b、将步骤a中进行表皮消毒处理后的水蛭转移至超清工作台内，并将其放入盛放有无菌PBS的无菌培养皿内，用无菌剪刀和镊子剪去蛭体两端取头部组织；b. Transfer the leech after the epidermal disinfection treatment in step a to the ultra-clear workbench, put it into a sterile petri dish containing sterile PBS, and cut off the two leech bodies with sterile scissors and tweezers. Take the head tissue; c、将步骤b中的头部组织转移到另一个装有PBS缓冲液的无菌培养皿内，浸过蛭体；将头部组织用无菌手术刀剖开头部组织，并在取表皮中间的组织后用无菌PBS缓冲液冲洗；c. Transfer the head tissue in step b to another sterile petri dish containing PBS buffer, and soak the leech body; cut the head tissue with a sterile scalpel, and cut the head tissue in the middle of the epidermis. Rinse with sterile PBS buffer after tissue removal; d、将步骤c中的组织置于75%的酒精内浸泡1min后，取出后用无菌PBS缓冲液漂洗，重复三次；d. After soaking the tissue in step c in 75% alcohol for 1 min, take it out and rinse with sterile PBS buffer, repeating three times; e、将步骤d中的组织置于盛放有无菌PBS的无菌培养皿内，用无菌剪刀和镊子剪碎成小组织块，并浸泡5min；弃掉漂浮在上部的组织碎屑，得到剪碎后的水蛭唾液腺组织块，剪碎后组织块的大小为1mm3；e. Place the tissue in step d in a sterile petri dish containing sterile PBS, cut it into small tissue pieces with sterile scissors and tweezers, and soak for 5min; discard the tissue debris floating on the upper part, Obtain the cut leech salivary gland tissue block, and the size of the cut tissue block is 1 mm 3 ; 所用的Insect-CCulture 昆虫细胞培养基基配方如下：The Insect-CCulture Insect Cell Culture Base formulation used was as follows: 无机盐：Inorganic salts: NaCl1420mg/L，ZnCL2 876.54mg/L，MgSO4·7H2O1743.48mg/L，KCl2080mg/L，KH2PO4·2H2O 1000mg/L；NaCl 1420mg/L, ZnCL 2 876.54mg/L, MgSO 4 7H 2 O 1743.48mg/L, KCl 2080mg/L, KH2PO 4 2H 2 O 1000mg/L; 氨基酸：Amino Acids: DL-丝氨酸9000mg/L，甘氨酸620mg/L，L-精氨酸盐酸盐700mg/L，L-天冬酰胺400mg/L，L-天冬氨酸350mg/L，L-半胱氨酸二盐酸盐160mg/L，L-谷氨酸3500mg/L，L-组氨酸盐酸盐2000mg/L，L-异亮氨酸200mg/L，L-亮氨酸150mg/L，L-赖氨酸盐酸盐850mg/L，L-甲硫氨酸100mg/L，L-苯丙氨酸100mg/L，L-脯氨酸500mg/L，L-苏氨酸200mg/L，L-色氨酸150mg/L，L-酪氨酸磷酸氢二钠580mg/L，L-缬氨酸200mg/L，ß-丙氨酸400mg/L，L-丙氨酸225mg/L；DL-serine 9000mg/L, glycine 620mg/L, L-arginine hydrochloride 700mg/L, L-asparagine 400mg/L, L-aspartic acid 350mg/L, L-cysteine Hydrochloride 160mg/L, L-glutamic acid 3500mg/L, L-histidine hydrochloride 2000mg/L, L-isoleucine 200mg/L, L-leucine 150mg/L, L-lysine Amino acid hydrochloride 850mg/L, L-methionine 100mg/L, L-phenylalanine 100mg/L, L-proline 500mg/L, L-threonine 200mg/L, L-color Amino acid 150mg/L, L-tyrosine disodium hydrogen phosphate 580mg/L, L-valine 200mg/L, ß-alanine 400mg/L, L-alanine 225mg/L; 维生素：Vitamins: D-生物素0.15mg/L，D-泛酸钙0.15mg/L，叶酸盐0.15mg/L，氰钴胺0.10mg/L，烟酸0.15mg/L，盐酸吡哆胺0.15mg/L，核黄素0.15mg/L，硫胺素0.10mg/L，对氨基苯甲酸0.15mg/L；D-biotin 0.15mg/L, D-calcium pantothenate 0.15mg/L, folate 0.15mg/L, cyanocobalamin 0.10mg/L, niacin 0.15mg/L, pyridoxamine hydrochloride 0.15mg/L, Riboflavin 0.15mg/L, thiamine 0.10mg/L, p-aminobenzoic acid 0.15mg/L; 其它：other: 蔗糖2000mg/L，D-葡萄糖1500mg/L，D-果糖500mg/L，麦芽糖600mg/L，α-酮戊二酸330mg/L，富马酸90mg/L，琥珀酸90mg/L，L-苹果酸90mg/L；Sucrose 2000mg/L, D-glucose 1500mg/L, D-fructose 500mg/L, maltose 600mg/L, α-ketoglutarate 330mg/L, fumaric acid 90mg/L, succinic acid 90mg/L, L-apple Acid 90mg/L; pH用1.0N KOH调节至7.4,使用前加入灭活的15%的昆虫专用特级胎牛血清;The pH was adjusted to 7.4 with 1.0N KOH, and inactivated 15% insect-specific special-grade fetal bovine serum was added before use; 步骤2）中所述组织块回收法的操作过程为：取出步骤1）的培养瓶，用吸管吸除瓶底多余液体，使用移液枪在瓶口缓慢加入4-6mL Insect-CCulture 昆虫细胞培养基，覆没组织块，继续原代培养，培养液颜色变深时及时更换新鲜培养液；培养6-12h后，待细胞将要铺满瓶底时，移除组织块，并将移除的组织块重新接种于新的培养瓶中，于培养箱中培养并及时更换新鲜培养液；The operation process of the tissue block recovery method described in step 2) is as follows: take out the culture bottle in step 1), suck off the excess liquid at the bottom of the bottle with a pipette, and slowly add 4-6mL of Insect-CCulture insect cell culture to the bottle mouth using a pipette. After culturing for 6-12 hours, when the cells are about to cover the bottom of the bottle, remove the tissue block and remove the tissue block. Re-inoculated in a new culture bottle, cultured in an incubator and replaced with fresh culture medium in time; 所述无菌PBS缓冲液内加入青霉素的含量为600U/mL，链霉素的含量为600U/mL，两性霉素B的含量为15μg/mL；The content of penicillin added to the sterile PBS buffer is 600 U/mL, the content of streptomycin is 600 U/mL, and the content of amphotericin B is 15 μg/mL; 步骤2）分离培养过程中的 Insect-CCulture 昆虫细胞培养基另加入青霉素的含量为600U/mL，链霉素的含量为600U/mL，两性霉素B的含量为15μg/mL，灭活的15%的昆虫专用特级胎牛血清；Step 2) Insect-CCulture insect cell culture medium during the isolation and culture process was additionally added with penicillin content of 600 U/mL, streptomycin content of 600 U/mL, amphotericin B content of 15 μg/mL, and inactivated 15 μg/mL. % special-grade fetal bovine serum for insects; 步骤3）所述胰酶消化法为：将步骤2）得到的细胞弃去培养液，经无菌PBS清洗后，加入2mL胰酶消化液，消化50s，轻轻晃动培养瓶，显微镜下观察，水蛭体细胞已有收缩但未浮起时，立即加入5mL Insect-CCulture 昆虫细胞培养基终止消化，轻轻晃动后弃去培养液，加入4mL PBS液，轻轻洗除残余的成纤维细胞后，加入2mL胰酶消化液，消化3min后，弃去1mL消化液，从底部拍打瓶底使细胞完全脱落，再加入5mLInsect-CCulture 昆虫细胞培养基终止消化，反复吹打细胞，使细胞团分散成单个细胞，利于培养；得到的细胞用八层纱布过滤后接种于含5mL Insect-CCulture 昆虫细胞培养基的25mL培养瓶内，培养温度25℃，培养时间3d。Step 3) The trypsin digestion method is as follows: discard the culture medium of the cells obtained in step 2), wash with sterile PBS, add 2 mL of trypsin digestion solution, digest for 50s, shake the culture bottle gently, and observe under a microscope. When the leech cells have shrunk but did not float, add 5 mL of Insect-CCulture insect cell culture medium immediately to terminate the digestion, shake gently and discard the culture medium, add 4 mL of PBS, and gently wash away the remaining fibroblasts. Add 2 mL of trypsin digestion solution, after 3 minutes of digestion, discard 1 mL of digestion solution, tap the bottom of the bottle from the bottom to completely fall off the cells, then add 5 mL of Insect-CCulture insect cell culture medium to terminate the digestion, and repeatedly pipet the cells to disperse the cell mass into single cells , which is conducive to cultivation; the obtained cells were filtered with eight layers of gauze and then inoculated into a 25 mL culture flask containing 5 mL of Insect-CCulture insect cell culture medium at a culture temperature of 25° C. and a culture time of 3 d.
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