CN110241068B - Method for removing phenol red in cell culture waste liquid - Google Patents
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Abstract
The invention discloses a method for removing phenol red in cell culture waste liquid, which is characterized by comprising the following steps: (1) performing ultrafiltration concentration on the cell culture waste liquid by using an ultrafiltration membrane package with 3-5 KD; (2) adding buffer solution or ultrapure water into the cell culture waste liquid after ultrafiltration concentration for washing and filtering; (3) adding Triton X-114 into the washed and filtered cell culture waste liquid to ensure that the volume concentration of the Triton X-114 is 1-5%, and preserving the heat at 4 ℃ for 30-60 min; (4) and (4) placing the substance obtained in the step (3) at 37-40 ℃ for further heat preservation for 30-60min, and finally centrifuging to remove the precipitate. The method for removing phenol red in the cell culture waste liquid has the advantages of excellent removal effect, simple removal method and no influence on effective components in the cell culture waste liquid.
Description
Technical Field
The invention belongs to the technical field of cell biology, and particularly relates to a method for removing phenol red in cell culture waste liquid.
Background
The culture medium left after the cells are cultured by the culture medium can be called as cell culture waste liquid, most researchers can pour the cell culture waste liquid, but the cell culture waste liquid also contains a large amount of nutrient substances and growth factors, so that the cell culture waste liquid has a good application value in recycling. However, when the cell culture waste liquid is recovered, if phenol red exists in the cell culture waste liquid, on one hand, the pH indicating effect is changed due to the change of the concentration of the phenol red; on the other hand, phenol red also acts as an estrogen, and when cells are cultured using the recovered cell culture solution, it adversely affects the cells. Therefore, it is necessary to remove phenol red from the cell culture waste liquid.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides a method for removing phenol red in cell culture waste liquid, which is simple and can effectively remove the phenol red in the cell culture waste liquid.
In order to achieve the purpose, the technical scheme adopted by the invention for solving the technical problems is as follows:
a method for removing phenol red in cell culture waste liquid comprises the following steps:
(1) ultrafiltering the cell culture waste liquid with ultrafiltration membrane of 3-5KD to concentrate to 1/2-2/3;
(2) adding buffer solution or ultrapure water with the volume of 2-4 times into the cell culture waste liquid after ultrafiltration concentration for washing and filtering;
(3) adding Triton X-114 into the washed and filtered cell culture waste liquid to ensure that the volume concentration of the Triton X-114 is 1-5%, and preserving the heat at 4 ℃ for 30-60 min;
(4) and (4) placing the substance obtained in the step (3) at 37-40 ℃ for further heat preservation for 30-60min, and finally centrifuging to remove the precipitate.
Further, in the step (2), a buffer solution or ultrapure water of 3 times volume is added to the cell culture waste liquid after the concentration by ultrafiltration.
Further, the buffer solution in step (2) is a neutral buffer solution, preferably a PBS buffer solution or a 0.5mol/L phosphate buffer solution.
Further, the volume concentration of Triton X-114 added in step (3) was 5%.
Further, in step (3), the temperature is maintained at 4 ℃ for 60 min.
Further, in the step (4), the substance obtained in the step (3) is placed at 37 ℃ and is kept for heat preservation for 60 min.
The method for removing phenol red in the cell culture waste liquid provided by the invention has the following technical effects:
phenol red is a small molecular substance, a part of phenol red can be removed by performing ultrafiltration concentration on cell culture waste liquid through an ultrafiltration membrane of 3-5KD, then buffer solution is used for washing and filtering the cell culture waste liquid, the amount of the buffer solution cannot be too much, so that the loss of effective components in the cell culture waste liquid is too much, if the amount of the buffer solution is too little, the residual amount of the phenol red cannot be well removed, and therefore the balance between the reduction of the residual amount of the phenol red and the control of the loss of the effective components is needed by the amount of the buffer solution, and the buffer solution with 3 times of volume is used for washing and filtering, so that the higher protein recovery rate can be ensured, and the residual amount of the phenol red can be reduced to a; when a lot of phenol red solution still exists in the cell culture waste liquid after washing and filtering, Triton X-114 is adopted for removing at the moment, Triton X-114 can be mixed and dissolved with water at 4 ℃, the solubility of Triton X-114 and phenol red is higher at low temperature of 4 ℃, the phenol red is compatible with Triton X-114 and is dissolved in water according to the principle of matter similarity compatibility, the solubility of Triton X-114 is reduced at 37 ℃, Triton X-114 is precipitated in a lower layer organic phase together with the phenol red, and the phenol red can be effectively removed through centrifugation. In addition, Triton X-114 has little effect on protein activity, so phenol red is removed without affecting the activity of effective substances in cell culture waste liquid.
Detailed Description
Example 1
A method for removing phenol red in cell culture waste liquid comprises the following steps:
(1) transferring the cell culture waste liquid into a sterile container rapidly according to aseptic operation in the process of stem cell culture passage and liquid change, sealing and storing in a refrigerator at 4 ℃, wherein the storage time is not more than 30 days;
(2) collecting waste liquid more than 600ml, taking out the sterile container filled with the cell culture waste liquid from the refrigerator, opening a bottle cap, pouring the sterile container into a liquid material cup, wherein the volume of the cell culture waste liquid in the liquid material cup is 600ml, connecting devices such as an ultrafiltration membrane package (or an ultrafiltration tube) with a molecular interception amount of 3KD, the liquid material cup, a pressure gauge and the like by using a No. 16 peristaltic pump hose, then starting the peristaltic pump to enable the pressure gauge to display a pressure of 30Bar, and closing the peristaltic pump when the volume of the cell culture waste liquid in the liquid material cup is reduced to half of the original volume (300 ml). Since phenol red is a small molecule substance, part of phenol red can be removed by ultrafiltration concentration.
(3) Adding 900ml PBS buffer solution into the cell culture waste liquid after ultrafiltration concentration, simultaneously connecting devices such as an ultrafiltration membrane package (or an ultrafiltration tube) with a molecular cut-off of 3KD, a feed liquid cup, a pressure gauge and the like by using a No. 16 peristaltic pump hose, then starting the peristaltic pump to ensure that the pressure gauge displays a pressure of 30Bar, and closing the peristaltic pump when the volume of the cell culture waste liquid in the feed liquid cup is reduced to half of the original volume (600 ml).
(4) And (3) adding Triton X-114 into the cell culture waste liquid obtained in the step (3) to ensure that the volume concentration of the Triton X-114 is 5%, carrying out shaking treatment in a water bath shaker at 4 ℃ for 1h, and immediately placing the shaking treatment in a water bath kettle at 37 ℃ for standing for 1 h.
(5) And (4) taking out the product obtained in the step (4), pouring the product into a centrifuge tube, centrifuging the product at room temperature at 10000r/min for 30min, separating the cell culture waste liquid into an upper liquid state and a lower gel state, carefully sucking out the split-phase supernatant, and obtaining the supernatant which is the cell culture waste liquid after phenol red removal.
Example 2
A method for removing phenol red in cell culture waste liquid comprises the following steps:
(1) transferring the cell culture waste liquid into a sterile container rapidly according to aseptic operation in the process of stem cell culture passage and liquid change, sealing and storing in a refrigerator at 4 ℃, wherein the storage time is not more than 30 days;
(2) collecting waste liquid more than 600ml, taking out the sterile container filled with the cell culture waste liquid from the refrigerator, opening a bottle cap, pouring the sterile container into a liquid material cup, wherein the volume of the cell culture waste liquid in the liquid material cup is 600ml, connecting devices such as an ultrafiltration membrane package (or an ultrafiltration tube) with a molecular interception amount of 3KD, the liquid material cup, a pressure gauge and the like by using a No. 16 peristaltic pump hose, then starting the peristaltic pump to enable the pressure gauge to display a pressure of 30Bar, and closing the peristaltic pump when the volume of the cell culture waste liquid in the liquid material cup is reduced to half of the original volume (300 ml). Since phenol red is a small molecule substance, part of phenol red can be removed by ultrafiltration concentration.
(3) Adding 900ml of 0.5mol/L phosphate buffer solution into the cell culture waste liquid after ultrafiltration concentration, simultaneously connecting an ultrafiltration membrane pack (or an ultrafiltration tube) with a molecular cut-off of 3KD, a feed liquid cup, a pressure gauge and other devices by using a No. 16 peristaltic pump hose, then starting the peristaltic pump to ensure that the pressure gauge displays 30Bar, and closing the peristaltic pump when the volume of the cell culture waste liquid in the feed liquid cup is reduced to half of the original volume (600 ml).
(4) And (3) adding Triton X-114 into the cell culture waste liquid obtained in the step (3) to enable the volume concentration of the Triton X-114 to be 1%, carrying out shaking treatment in a water bath shaker at 4 ℃ for 1h, and immediately placing the shaking treatment in a water bath kettle at 40 ℃ for standing for 1 h.
(5) And (4) taking out the product obtained in the step (4), pouring the product into a centrifuge tube, centrifuging at 12000r/min for 15min at room temperature, separating the cell culture waste liquid into an upper liquid state and a lower gel state, carefully sucking out the split-phase supernatant, and obtaining the supernatant which is the cell culture waste liquid after phenol red is removed.
Example 3
A method for removing phenol red in cell culture waste liquid comprises the following steps:
(1) transferring the cell culture waste liquid into a sterile container rapidly according to aseptic operation in the process of stem cell culture passage and liquid change, sealing and storing in a refrigerator at 4 ℃, wherein the storage time is not more than 30 days;
(2) collecting waste liquid more than 600ml, taking out the sterile container filled with the cell culture waste liquid from the refrigerator, opening a bottle cap, pouring the sterile container into a liquid material cup, wherein the volume of the cell culture waste liquid in the liquid material cup is 600ml, connecting devices such as an ultrafiltration membrane package (or an ultrafiltration tube) with a molecular interception amount of 3KD, the liquid material cup, a pressure gauge and the like by using a No. 16 peristaltic pump hose, then starting the peristaltic pump to enable the pressure gauge to display a pressure of 30Bar, and closing the peristaltic pump when the volume of the cell culture waste liquid in the liquid material cup is reduced to half of the original volume (300 ml). Since phenol red is a small molecule substance, part of phenol red can be removed by ultrafiltration concentration.
(3) Adding 900ml of water into the cell culture waste liquid after ultrafiltration concentration, simultaneously connecting an ultrafiltration membrane pack (or an ultrafiltration tube) with a molecular cut-off of 3KD, a feed liquid cup, a pressure gauge and the like by using a No. 16 peristaltic pump hose, then starting the peristaltic pump to ensure that the pressure gauge displays a pressure of 30Bar, and closing the peristaltic pump when the volume of the cell culture waste liquid in the feed liquid cup is reduced to half of the original volume (600 ml).
(4) And (4) adding Triton X-114 into the cell culture waste liquid obtained in the step (3) to enable the volume concentration of the Triton X-114 to be 3%, carrying out oscillation treatment in a water bath shaker at 4 ℃ for 30min, and immediately placing the shaking table into a water bath kettle at 40 ℃ for standing for 30 min.
(5) And (4) taking out the product obtained in the step (4), pouring the product into a centrifuge tube, centrifuging at 12000r/min for 15min at room temperature, separating the cell culture waste liquid into an upper liquid state and a lower gel state, carefully sucking out the split-phase supernatant, and obtaining the supernatant which is the cell culture waste liquid after phenol red is removed.
Example 4
A method for removing phenol red in cell culture waste liquid comprises the following steps:
(1) transferring the cell culture waste liquid into a sterile container rapidly according to aseptic operation in the process of stem cell culture passage and liquid change, sealing and storing in a refrigerator at 4 ℃, wherein the storage time is not more than 30 days;
(2) collecting waste liquid more than 600ml, taking out the sterile container filled with the cell culture waste liquid from the refrigerator, opening a bottle cap, pouring the sterile container into a liquid material cup, wherein the volume of the cell culture waste liquid in the liquid material cup is 600ml, connecting devices such as an ultrafiltration membrane package (or an ultrafiltration tube) with a molecular interception amount of 3KD, the liquid material cup, a pressure gauge and the like by using a No. 16 peristaltic pump hose, then starting the peristaltic pump to enable the pressure gauge to display a pressure of 30Bar, and closing the peristaltic pump when the volume of the cell culture waste liquid in the liquid material cup is reduced to half of the original volume (300 ml). Since phenol red is a small molecule substance, part of phenol red can be removed by ultrafiltration concentration.
(3) Adding 900ml PBS buffer solution into the cell culture waste liquid after ultrafiltration concentration, simultaneously connecting devices such as an ultrafiltration membrane package (or an ultrafiltration tube) with a molecular cut-off of 3KD, a feed liquid cup, a pressure gauge and the like by using a No. 16 peristaltic pump hose, then starting the peristaltic pump to ensure that the pressure gauge displays a pressure of 30Bar, and closing the peristaltic pump when the volume of the cell culture waste liquid in the feed liquid cup is reduced to half of the original volume (600 ml).
(4) And (4) adding Triton X-114 into the cell culture waste liquid obtained in the step (3) to enable the volume concentration of the Triton X-114 to be 2%, carrying out shaking treatment in a water bath shaker at 4 ℃ for 50min, and immediately placing the shaking treatment in a water bath kettle at 40 ℃ for standing for 40 min.
(5) And (4) taking out the product obtained in the step (4), pouring the product into a centrifuge tube, centrifuging the product at room temperature at 10000r/min for 30min, separating the cell culture waste liquid into an upper liquid state and a lower gel state, carefully sucking out the split-phase supernatant, and obtaining the supernatant which is the cell culture waste liquid after phenol red removal.
Example 5
A method for removing phenol red in cell culture waste liquid comprises the following steps:
(1) transferring the cell culture waste liquid into a sterile container rapidly according to aseptic operation in the process of stem cell culture passage and liquid change, sealing and storing in a refrigerator at 4 ℃, wherein the storage time is not more than 30 days;
(2) collecting waste liquid more than 600ml, taking out the sterile container filled with the cell culture waste liquid from the refrigerator, opening a bottle cap, pouring the sterile container into a liquid material cup, wherein the volume of the cell culture waste liquid in the liquid material cup is 600ml, connecting devices such as an ultrafiltration membrane package (or an ultrafiltration tube) with a molecular interception amount of 3KD, the liquid material cup, a pressure gauge and the like by using a No. 16 peristaltic pump hose, then starting the peristaltic pump to enable the pressure gauge to display a pressure of 30Bar, and closing the peristaltic pump when the volume of the cell culture waste liquid in the liquid material cup is reduced to half of the original volume (300 ml). Since phenol red is a small molecule substance, part of phenol red can be removed by ultrafiltration concentration.
(3) Adding 900ml PBS buffer solution into the cell culture waste liquid after ultrafiltration concentration, simultaneously connecting devices such as an ultrafiltration membrane package (or an ultrafiltration tube) with a molecular cut-off of 3KD, a feed liquid cup, a pressure gauge and the like by using a No. 16 peristaltic pump hose, then starting the peristaltic pump to ensure that the pressure gauge displays a pressure of 30Bar, and closing the peristaltic pump when the volume of the cell culture waste liquid in the feed liquid cup is reduced to half of the original volume (600 ml).
(4) And (3) adding Triton X-114 into the cell culture waste liquid obtained in the step (3) to enable the volume concentration of the Triton X-114 to be 4%, carrying out shaking treatment in a water bath shaker at 4 ℃ for 40min, and immediately placing the shaking treatment in a water bath kettle at 37 ℃ for standing for 30 min.
(5) And (4) taking out the product obtained in the step (4), pouring the product into a centrifuge tube, centrifuging the product at room temperature at 10000r/min for 30min, separating the cell culture waste liquid into an upper liquid state and a lower gel state, carefully sucking out the split-phase supernatant, and obtaining the supernatant which is the cell culture waste liquid after phenol red removal.
Comparative example 1
A method for removing phenol red in cell culture waste liquid comprises the following steps:
(1) transferring the cell culture waste liquid into a sterile container rapidly according to aseptic operation in the process of stem cell culture passage and liquid change, sealing and storing in a refrigerator at 4 ℃, wherein the storage time is not more than 30 days;
(2) collecting waste liquid to more than 600ml, adding Triton X-114 into the cell culture waste liquid to make the volume concentration of Triton X-114 to be 5%, shaking in a water bath shaker at 4 ℃ for 1h, and immediately placing in a water bath kettle at 37 ℃ for 1 h.
(3) Taking out the product obtained in the step (2), pouring the product into a centrifuge tube, centrifuging the product at room temperature at 10000r/min for 30min, separating the cell culture waste liquid into an upper liquid state and a lower gel state, and carefully sucking out the split-phase supernatant;
(4) taking 600ml of the supernatant obtained in the step (3), connecting devices such as an ultrafiltration membrane package (or an ultrafiltration tube) with a molecular interception of 3KD, a feed liquid cup, a pressure gauge and the like by using a No. 16 peristaltic pump hose, then starting the peristaltic pump to enable the pressure gauge to display a pressure of 30Bar, and closing the peristaltic pump when the volume of the cell culture waste liquid in the feed liquid cup is reduced to half of the original volume (300 ml).
(4) Adding 900ml PBS buffer solution into the cell culture waste liquid after ultrafiltration concentration, simultaneously connecting devices such as an ultrafiltration membrane package (or an ultrafiltration tube) with a molecular cut-off of 3KD, a feed liquid cup, a pressure gauge and the like by using a No. 16 peristaltic pump hose, then starting the peristaltic pump to ensure that the pressure gauge displays a pressure of 30Bar, and closing the peristaltic pump when the volume of the cell culture waste liquid in the feed liquid cup is reduced to half of the original volume (600 ml).
Comparative example 2
A method for removing phenol red in cell culture waste liquid comprises the following steps:
(1) transferring the cell culture waste liquid into a sterile container rapidly according to aseptic operation in the process of stem cell culture passage and liquid change, sealing and storing in a refrigerator at 4 ℃, wherein the storage time is not more than 30 days;
(2) collecting waste liquid more than 600ml, taking out the sterile container filled with the cell culture waste liquid from the refrigerator, opening a bottle cap, pouring the sterile container into a liquid material cup, wherein the volume of the cell culture waste liquid in the liquid material cup is 600ml, connecting devices such as an ultrafiltration membrane package (or an ultrafiltration tube) with a molecular interception amount of 3KD, the liquid material cup, a pressure gauge and the like by using a No. 16 peristaltic pump hose, then starting the peristaltic pump to enable the pressure gauge to display a pressure of 30Bar, and closing the peristaltic pump when the volume of the cell culture waste liquid in the liquid material cup is reduced to half of the original volume (300 ml). Since phenol red is a small molecule substance, part of phenol red can be removed by ultrafiltration concentration.
(3) Adding Triton X-114 into the cell culture waste liquid after ultrafiltration concentration to ensure that the volume concentration of the Triton X-114 is 5%, shaking in a water bath shaker at 4 ℃ for 1h, and immediately placing in a water bath kettle at 37 ℃ for 1 h.
(4) And (4) taking out the product obtained in the step (3), pouring the product into a centrifuge tube, centrifuging the product at room temperature at 10000r/min for 30min, separating the cell culture waste liquid into an upper liquid state and a lower gel state, carefully sucking out the split-phase supernatant, and obtaining the supernatant which is the cell culture waste liquid after phenol red removal.
The phenol red removing effect in examples 1 to 5 and comparative examples 1 to 2 was examined:
1. color observation
The cell culture waste liquid before removal of phenol red was red, and after removal of phenol red, the cell culture waste liquid in each of examples 1 to 5 and comparative examples 1 to 2 was pale yellow.
2. Phenol red content detection
The method for measuring the content of phenol red by adopting a dual-wavelength spectrophotometry comprises the following steps: sucking 0.5ml of a sample to be detected, adding 4.5ml of 0.1mol/L NaOH solution, uniformly mixing, taking 0.1mol/L NaOH solution as a reference, measuring absorbance at 558nm and 570nm, and then calculating phenol red concentration, wherein the result is shown in the following table:
| before removal | After being removed | |
| Example 1 | 28.108mg/L | 1.357mg/L |
| Example 2 | 28.108mg/L | 2.052mg/L |
| Example 3 | 28.108mg/L | 1.953mg/L |
| Example 4 | 28.108mg/L | 2.022mg/L |
| Example 5 | 28.108mg/L | 1.816mg/L |
| Comparative example 1 | 28.108mg/L | 5.618mg/L |
| Comparative example 2 | 28.108mg/L | 9.215mg/L |
3. Protein concentration detection
The method for measuring the protein concentration of the cell culture waste liquid by adopting an ultraviolet spectrophotometry (or BCA detection method) comprises the following specific steps: 3ml of the sample to be tested was aspirated, the absorbance was measured at 260nm and 280nm using a 0.9% NaCl solution as a reference, and then the protein concentration was calculated, the results of which are shown in the following Table:
| before removal | After being removed | |
| Example 1 | 260.319mg/L | 226.458mg/L |
| Example 2 | 260.319mg/L | 214.254mg/L |
| Example 3 | 260.319mg/L | 213.531mg/L |
| Example 4 | 260.319mg/L | 219.069mg/L |
| Example 5 | 260.319mg/L | 218.163mg/L |
| Comparative example 1 | 260.319mg/L | 208.018mg/L |
| Comparative example 2 | 260.319mg/L | 242.019mg/L |
As is clear from the above, phenol red was removed only by the method of the present invention according to the steps of the present invention, which is excellent in removal effect, simple in removal method, and free from influence on the effective components in the cell culture waste liquid.
Claims (6)
1. A method for removing phenol red in cell culture waste liquid is characterized by comprising the following steps:
(1) ultrafiltering the cell culture waste liquid with ultrafiltration membrane of 3-5KD to concentrate to 1/2-2/3;
(2) adding buffer solution or ultrapure water with the volume of 2-4 times into the cell culture waste liquid after ultrafiltration concentration for washing and filtering;
(3) adding Triton X-114 into the washed and filtered cell culture waste liquid to ensure that the volume concentration of the Triton X-114 is 1-5%, and preserving the heat at 4 ℃ for 30-60 min;
(4) and (4) placing the substance obtained in the step (3) at 37-40 ℃ for further heat preservation for 30-60min, and finally centrifuging to remove the precipitate.
2. The method for removing phenol red from a cell culture waste liquid according to claim 1, wherein a buffer or ultrapure water is added to the cell culture waste liquid after the concentration by ultrafiltration in the step (2) in a volume of 3 times.
3. The method for removing phenol red from a cell culture waste solution according to claim 1 or 2, wherein the buffer in the step (2) is a neutral buffer.
4. The method for removing phenol red from a cell culture waste solution according to claim 1, wherein the volume concentration of Triton X-114 added in the step (3) is 5%.
5. The method for removing phenol red from a cell culture waste liquid according to claim 1, wherein the temperature in step (3) is maintained at 4 ℃ for 60 min.
6. The method for removing phenol red from a cell culture waste liquid according to claim 1, wherein in the step (4), the product obtained in the step (3) is kept at 37 ℃ for further incubation for 60 min.
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