CN110241068A - A method of it is phenol red in removal cell culture waste liquid - Google Patents
A method of it is phenol red in removal cell culture waste liquid Download PDFInfo
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Abstract
The invention discloses a kind of methods phenol red in removal cell culture waste liquid, which comprises the following steps: (1) cell culture waste liquid is concentrated by ultrafiltration through the ultrafiltration membrane packet of 3-5KD；(2) buffer is added into the cell culture waste liquid after ultrafiltration concentration or ultrapure water carries out filter wash；(3) Triton X-114 is added into the cell culture waste liquid after filter wash, so that the volumetric concentration of Triton X-114 is 1-5%, in 4 DEG C of heat preservation 30-60min；(4) step (3) gains are placed in 37-40 DEG C to continue to keep the temperature 30-60min, are finally centrifuged, removal precipitating.Phenol red in removal cell culture waste liquid by the method for the invention, removal effect is excellent, and minimizing technology is simple, and will not impact to effective component in cell culture waste liquid.
Description
Technical field
The invention belongs to technical field of cell biology, and in particular to a kind of to remove side phenol red in cell culture waste liquid
Method.
Background technique
Cell culture waste liquid can be referred to as using remaining culture medium after the complete cell of culture medium culture, most researchers can incite somebody to action
Cell culture waste liquid is outwelled, but also contains a large amount of nutriment and growth factor in cell culture waste liquid, therefore is trained to cell
Feeding waste liquid is recycled with good application value.But when recycling cell culture waste liquid, in cell culture waste liquid such as
For fruit there are phenol red, variation on the one hand can have occurred because of phenol red concentration causes pH indicating effect to be changed；It on the other hand is phenol
It is red also to play the role of estrogen, when using cell culture fluid culture cell after the recovery, unfavorable shadow can be generated to cell
It rings.Therefore, remove cell culture waste liquid in it is phenol red be necessary.
Summary of the invention
For above-mentioned deficiency in the prior art, the present invention provides sides phenol red in a kind of removal cell culture waste liquid
Method, this method is simple, can effectively remove phenol red in cell waste liquid.
To achieve the above object, the technical solution adopted by the present invention to solve the technical problems is:
A method of it is phenol red in removal cell culture waste liquid, comprising the following steps:
(1) cell culture waste liquid is concentrated by ultrafiltration through the ultrafiltration membrane packet of 3-5KD to the 1/2-2/3 of original volume；
(2) buffer or ultrapure water that 2-4 times of volume is added into the cell culture waste liquid after ultrafiltration concentration carry out filter wash；
(3) Triton X-114 is added into the cell culture waste liquid after filter wash, so that the volume of Triton X-114 is dense
Degree is 1-5%, in 4 DEG C of heat preservation 30-60min；
(4) step (3) gains are placed in 37-40 DEG C to continue to keep the temperature 30-60min, are finally centrifuged, removal precipitating.
Further, the buffer or super of 3 times of volumes is added in step (2) into the cell culture waste liquid after ultrafiltration concentration
Pure water.
Further, step (2) buffer is neutral buffered liquid, and the preferably phosphoric acid of PBS buffer solution or 0.5mol/L is slow
Fliud flushing.
Further, the volumetric concentration for the Triton X-114 being added in step (3) is 5%.
Further, in 4 DEG C of heat preservation 60min in step (3).
Further, step (3) gains 37 DEG C are placed in step (4) to continue to keep the temperature 60min.
Phenol red method, has following technical effect that in removal cell culture waste liquid provided by the invention
Phenol red is small-molecule substance, and be concentrated by ultrafiltration through the ultrafiltration membrane packet of 3-5KD by cell culture waste liquid first can be with
It removes a part of phenol red, filter wash then is carried out to cell culture waste liquid with buffer, the amount of buffer cannot be excessive, can excessively make
The effective component obtained in cell culture waste liquid is lost excessively, if the amount of buffer is very little, cannot remove phenol red residual well
Amount makes in the application so the amount of buffer needs to balance between the phenol red residual quantity of reduction and control effective component are lost
Filter wash is carried out with the buffer of 3 times of volumes, not only can guarantee higher protein recovery, but also can largely reduce phenol red residual
Amount；Still there are many phenol red solutions in cell culture waste liquid after filter wash, be removed at this time using Triton X-114,
Triton X-114 is water-miscible under the conditions of 4 DEG C, and Triton X-114 and phenol red solubility is all under the conditions of 4 DEG C of low temperature
Higher, according to substance similar compatibility principle, phenol red meeting is compatible with Triton X-114, and is dissolved in water, and at 37 DEG C,
Triton X-114 solubility becomes smaller, and Triton X-114 is precipitated in lower layer's organic phase together in conjunction with phenol red, by being centrifuged just
It can effectively remove phenol red.In addition, Triton X-114 on protein active influence very little, therefore remove it is phenol red while will not be right
The activity of active principle impacts in cell culture waste liquid.
Specific embodiment
Embodiment 1
A method of it is phenol red in removal cell culture waste liquid, comprising the following steps:
(1) in stem cell subculture and by sterile working quickly cell culture waste liquid is transferred to during change liquid sterile
In container, it is sealed in 4 DEG C of refrigerators, the holding time is no more than 30 days；
(2) it collects waste liquid and reaches 600ml or more, the sterile chamber that cell culture waste liquid is housed is taken out out of refrigerator, open
Bottle cap pours into feed liquid cup, and the volume of cell culture waste liquid is 600ml in feed liquid cup, is connected using No. 16 wriggling pump hoses
Molecule interception is the devices such as ultrafiltration membrane packet (or super filter tube), feed liquid cup and the pressure gauge of 3KD, is then turned on peristaltic pump, makes
Pressure gauge shows that pressure is 30Bar, when feed liquid cup inner cell culture waste liquid volume is reduced to original volume half (300ml), closes
Close peristaltic pump.Because it is phenol red be small-molecule substance, be concentrated by ultrafiltration that can to remove part phenol red.
(3) 900ml PBS buffer solution is added in the cell culture waste liquid after ultrafiltration concentration, while using No. 16 peristaltic pumps
Hose connects the devices such as ultrafiltration membrane packet (or super filter tube), feed liquid cup and the pressure gauge that molecule interception is 3KD, is then turned on
Peristaltic pump makes pressure gauge show pressure 30Bar, when feed liquid cup inner cell culture waste liquid volume is reduced to original volume half
(600ml) closes peristaltic pump.
(4) Triton X-114 is added into the resulting cell culture waste liquid of step (3), so that the body of Triton X-114
Product concentration is 5%, and the oscillation treatment 1h in 4 DEG C of shaking baths is put into 37 DEG C of water-baths immediately after and stands 1h.
(5) step (4) gains are taken out, is poured into centrifuge tube, under room temperature, 10000r/min is centrifuged 30min, carefully
Born of the same parents cultivate waste liquid and are divided into supernatant liquid shape and lower layer's gel, careful to be sucked out split-phase supernatant, supernatant be remove it is phenol red after
Cell culture waste liquid.
Embodiment 2
A method of it is phenol red in removal cell culture waste liquid, comprising the following steps:
(1) in stem cell subculture and by sterile working quickly cell culture waste liquid is transferred to during change liquid sterile
In container, it is sealed in 4 DEG C of refrigerators, the holding time is no more than 30 days；
(2) it collects waste liquid and reaches 600ml or more, the sterile chamber that cell culture waste liquid is housed is taken out out of refrigerator, open
Bottle cap pours into feed liquid cup, and the volume of cell culture waste liquid is 600ml in feed liquid cup, is connected using No. 16 wriggling pump hoses
Molecule interception is the devices such as ultrafiltration membrane packet (or super filter tube), feed liquid cup and the pressure gauge of 3KD, is then turned on peristaltic pump, makes
Pressure gauge shows that pressure is 30Bar, when feed liquid cup inner cell culture waste liquid volume is reduced to original volume half (300ml), closes
Close peristaltic pump.Because it is phenol red be small-molecule substance, be concentrated by ultrafiltration that can to remove part phenol red.
(3) phosphate buffer of 900ml 0.5mol/L is added in the cell culture waste liquid after ultrafiltration concentration, makes simultaneously
Ultrafiltration membrane packet (or super filter tube), feed liquid cup and the pressure gauge etc. that molecule interception is 3KD are connected with No. 16 wriggling pump hoses
Device is then turned on peristaltic pump, so that pressure gauge is shown pressure 30Bar, is reduced to feed liquid cup inner cell culture waste liquid volume
When original volume half (600ml), peristaltic pump is closed.
(4) Triton X-114 is added into the resulting cell culture waste liquid of step (3), so that the body of Triton X-114
Product concentration is 1%, and the oscillation treatment 1h in 4 DEG C of shaking baths is put into 40 DEG C of water-baths immediately after and stands 1h.
(5) step (4) gains are taken out, is poured into centrifuge tube, under room temperature, 12000r/min is centrifuged 15min, carefully
Born of the same parents cultivate waste liquid and are divided into supernatant liquid shape and lower layer's gel, careful to be sucked out split-phase supernatant, supernatant be remove it is phenol red after
Cell culture waste liquid.
Embodiment 3
A method of it is phenol red in removal cell culture waste liquid, comprising the following steps:
(1) in stem cell subculture and by sterile working quickly cell culture waste liquid is transferred to during change liquid sterile
In container, it is sealed in 4 DEG C of refrigerators, the holding time is no more than 30 days；
(2) it collects waste liquid and reaches 600ml or more, the sterile chamber that cell culture waste liquid is housed is taken out out of refrigerator, open
Bottle cap pours into feed liquid cup, and the volume of cell culture waste liquid is 600ml in feed liquid cup, is connected using No. 16 wriggling pump hoses
Molecule interception is the devices such as ultrafiltration membrane packet (or super filter tube), feed liquid cup and the pressure gauge of 3KD, is then turned on peristaltic pump, makes
Pressure gauge shows that pressure is 30Bar, when feed liquid cup inner cell culture waste liquid volume is reduced to original volume half (300ml), closes
Close peristaltic pump.Because it is phenol red be small-molecule substance, be concentrated by ultrafiltration that can to remove part phenol red.
(3) 900ml water is added in the cell culture waste liquid after ultrafiltration concentration, while being connected using No. 16 wriggling pump hoses
Good molecule interception is the devices such as ultrafiltration membrane packet (or super filter tube), feed liquid cup and the pressure gauge of 3KD, is then turned on peristaltic pump,
Pressure gauge is set to show pressure 30Bar, when feed liquid cup inner cell culture waste liquid volume is reduced to original volume half (600ml),
Close peristaltic pump.
(4) Triton X-114 is added into the resulting cell culture waste liquid of step (3), so that the body of Triton X-114
Product concentration is 3%, and the oscillation treatment 30min in 4 DEG C of shaking baths is put into 40 DEG C of water-baths immediately after and stands 30min.
(5) step (4) gains are taken out, is poured into centrifuge tube, under room temperature, 12000r/min is centrifuged 15min, carefully
Born of the same parents cultivate waste liquid and are divided into supernatant liquid shape and lower layer's gel, careful to be sucked out split-phase supernatant, supernatant be remove it is phenol red after
Cell culture waste liquid.
Embodiment 4
A method of it is phenol red in removal cell culture waste liquid, comprising the following steps:
(1) in stem cell subculture and by sterile working quickly cell culture waste liquid is transferred to during change liquid sterile
In container, it is sealed in 4 DEG C of refrigerators, the holding time is no more than 30 days；
(2) it collects waste liquid and reaches 600ml or more, the sterile chamber that cell culture waste liquid is housed is taken out out of refrigerator, open
Bottle cap pours into feed liquid cup, and the volume of cell culture waste liquid is 600ml in feed liquid cup, is connected using No. 16 wriggling pump hoses
Molecule interception is the devices such as ultrafiltration membrane packet (or super filter tube), feed liquid cup and the pressure gauge of 3KD, is then turned on peristaltic pump, makes
Pressure gauge shows that pressure is 30Bar, when feed liquid cup inner cell culture waste liquid volume is reduced to original volume half (300ml), closes
Close peristaltic pump.Because it is phenol red be small-molecule substance, be concentrated by ultrafiltration that can to remove part phenol red.
(3) 900ml PBS buffer solution is added in the cell culture waste liquid after ultrafiltration concentration, while using No. 16 peristaltic pumps
Hose connects the devices such as ultrafiltration membrane packet (or super filter tube), feed liquid cup and the pressure gauge that molecule interception is 3KD, is then turned on
Peristaltic pump makes pressure gauge show pressure 30Bar, when feed liquid cup inner cell culture waste liquid volume is reduced to original volume half
(600ml) closes peristaltic pump.
(4) Triton X-114 is added into the resulting cell culture waste liquid of step (3), so that the body of Triton X-114
Product concentration is 2%, and the oscillation treatment 50min in 4 DEG C of shaking baths is put into 40 DEG C of water-baths immediately after and stands 40min.
(5) step (4) gains are taken out, is poured into centrifuge tube, under room temperature, 10000r/min is centrifuged 30min, carefully
Born of the same parents cultivate waste liquid and are divided into supernatant liquid shape and lower layer's gel, careful to be sucked out split-phase supernatant, supernatant be remove it is phenol red after
Cell culture waste liquid.
Embodiment 5
A method of it is phenol red in removal cell culture waste liquid, comprising the following steps:
(1) in stem cell subculture and by sterile working quickly cell culture waste liquid is transferred to during change liquid sterile
In container, it is sealed in 4 DEG C of refrigerators, the holding time is no more than 30 days；
(2) it collects waste liquid and reaches 600ml or more, the sterile chamber that cell culture waste liquid is housed is taken out out of refrigerator, open
Bottle cap pours into feed liquid cup, and the volume of cell culture waste liquid is 600ml in feed liquid cup, is connected using No. 16 wriggling pump hoses
Molecule interception is the devices such as ultrafiltration membrane packet (or super filter tube), feed liquid cup and the pressure gauge of 3KD, is then turned on peristaltic pump, makes
Pressure gauge shows that pressure is 30Bar, when feed liquid cup inner cell culture waste liquid volume is reduced to original volume half (300ml), closes
Close peristaltic pump.Because it is phenol red be small-molecule substance, be concentrated by ultrafiltration that can to remove part phenol red.
(3) 900ml PBS buffer solution is added in the cell culture waste liquid after ultrafiltration concentration, while using No. 16 peristaltic pumps
Hose connects the devices such as ultrafiltration membrane packet (or super filter tube), feed liquid cup and the pressure gauge that molecule interception is 3KD, is then turned on
Peristaltic pump makes pressure gauge show pressure 30Bar, when feed liquid cup inner cell culture waste liquid volume is reduced to original volume half
(600ml) closes peristaltic pump.
(4) Triton X-114 is added into the resulting cell culture waste liquid of step (3), so that the body of Triton X-114
Product concentration is 4%, and the oscillation treatment 40min in 4 DEG C of shaking baths is put into 37 DEG C of water-baths immediately after and stands 30min.
(5) step (4) gains are taken out, is poured into centrifuge tube, under room temperature, 10000r/min is centrifuged 30min, carefully
Born of the same parents cultivate waste liquid and are divided into supernatant liquid shape and lower layer's gel, careful to be sucked out split-phase supernatant, supernatant be remove it is phenol red after
Cell culture waste liquid.
Comparative example 1
A method of it is phenol red in removal cell culture waste liquid, comprising the following steps:
(1) in stem cell subculture and by sterile working quickly cell culture waste liquid is transferred to during change liquid sterile
In container, it is sealed in 4 DEG C of refrigerators, the holding time is no more than 30 days；
(2) it collects waste liquid and reaches 600ml or more, Triton X-114 is added into cell culture waste liquid, so that Triton
The volumetric concentration of X-114 is 5%, and the oscillation treatment 1h in 4 DEG C of shaking baths is put into 37 DEG C of water-baths immediately after and stands
1h。
(3) step (2) gains are taken out, is poured into centrifuge tube, under room temperature, 10000r/min is centrifuged 30min, carefully
Born of the same parents cultivate waste liquid and are divided into supernatant liquid shape and lower layer's gel, careful that split-phase supernatant is sucked out；
(4) supernatant 600ml obtained by step (3) is taken, connecting molecule interception using No. 16 wriggling pump hoses is 3KD's
The devices such as ultrafiltration membrane packet (or super filter tube), feed liquid cup and pressure gauge, are then turned on peristaltic pump, and pressure gauge is made to show pressure
30Bar closes peristaltic pump when feed liquid cup inner cell culture waste liquid volume is reduced to original volume half (300ml).
(4) 900ml PBS buffer solution is added in the cell culture waste liquid after ultrafiltration concentration, while using No. 16 peristaltic pumps
Hose connects the devices such as ultrafiltration membrane packet (or super filter tube), feed liquid cup and the pressure gauge that molecule interception is 3KD, is then turned on
Peristaltic pump makes pressure gauge show pressure 30Bar, when feed liquid cup inner cell culture waste liquid volume is reduced to original volume half
(600ml) closes peristaltic pump.
Comparative example 2
A method of it is phenol red in removal cell culture waste liquid, comprising the following steps:
(1) in stem cell subculture and by sterile working quickly cell culture waste liquid is transferred to during change liquid sterile
In container, it is sealed in 4 DEG C of refrigerators, the holding time is no more than 30 days；
(2) it collects waste liquid and reaches 600ml or more, the sterile chamber that cell culture waste liquid is housed is taken out out of refrigerator, open
Bottle cap pours into feed liquid cup, and the volume of cell culture waste liquid is 600ml in feed liquid cup, is connected using No. 16 wriggling pump hoses
Molecule interception is the devices such as ultrafiltration membrane packet (or super filter tube), feed liquid cup and the pressure gauge of 3KD, is then turned on peristaltic pump, makes
Pressure gauge shows that pressure is 30Bar, when feed liquid cup inner cell culture waste liquid volume is reduced to original volume half (300ml), closes
Close peristaltic pump.Because it is phenol red be small-molecule substance, be concentrated by ultrafiltration that can to remove part phenol red.
(3) Triton X-114 is added into the cell culture waste liquid after ultrafiltration concentration, so that the body of Triton X-114
Product concentration is 5%, and the oscillation treatment 1h in 4 DEG C of shaking baths is put into 37 DEG C of water-baths immediately after and stands 1h.
(4) step (3) gains are taken out, is poured into centrifuge tube, under room temperature, 10000r/min is centrifuged 30min, carefully
Born of the same parents cultivate waste liquid and are divided into supernatant liquid shape and lower layer's gel, careful to be sucked out split-phase supernatant, supernatant be remove it is phenol red after
Cell culture waste liquid.
Detect phenol red removal effect in embodiment 1-5 and comparative example 1-2:
1, color observation
Remove it is phenol red before, cell culture waste liquid takes on a red color, remove it is phenol red after, cell training in embodiment 1-5 and comparative example 1-2
Feeding waste liquid becomes light yellow.
2, phenol red content detection
Using the phenol red content of Dual-Wavelength Spectrophotometric Determination, method particularly includes: sample to be tested 0.5ml is drawn, is added
The NaOH solution 4.5ml of 0.1mol/L is mixed, and using the NaOH solution of 0.1mol/L as reference, is surveyed at 558nm and 570nm
Determine absorbance, then calculate phenol red concentration, the result is shown in following tables:
| Before removal | After removal | |
| Embodiment 1 | 28.108mg/L | 1.357mg/L |
| Embodiment 2 | 28.108mg/L | 2.052mg/L |
| Embodiment 3 | 28.108mg/L | 1.953mg/L |
| Embodiment 4 | 28.108mg/L | 2.022mg/L |
| Embodiment 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 detects
Using the protein concentration of ultraviolet spectrophotometry (or BCA detection method) measurement cell culture waste liquid, specific side
Method are as follows: draw sample to be tested 3ml, using 0.9%NaCl solution as reference, absorbance is measured at 260nm and 280nm, then
Protein concentration is calculated, the result is shown in following tables:
| Before removal | After removal | |
| Embodiment 1 | 260.319mg/L | 226.458mg/L |
| Embodiment 2 | 260.319mg/L | 214.254mg/L |
| Embodiment 3 | 260.319mg/L | 213.531mg/L |
| Embodiment 4 | 260.319mg/L | 219.069mg/L |
| Embodiment 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 |
It can be seen from the above, only by the method for the invention, phenol red, removal effect is removed according to corresponding steps of the present invention
Excellent, minimizing technology is simple, and will not impact to effective component in cell culture waste liquid.
Claims (6)
1. a kind of phenol red method in removal cell culture waste liquid, which comprises the following steps:
(1) cell culture waste liquid is concentrated by ultrafiltration through the ultrafiltration membrane packet of 3-5KD to the 1/2-2/3 of original volume；
(2) buffer or ultrapure water that 2-4 times of volume is added into the cell culture waste liquid after ultrafiltration concentration carry out filter wash；
(3) Triton X-114 is added into the cell culture waste liquid after filter wash, so that the volumetric concentration of Triton X-114 is
1-5%, in 4 DEG C of heat preservation 30-60min；
(4) step (3) gains are placed in 37-40 DEG C to continue to keep the temperature 30-60min, are finally centrifuged, removal precipitating.
2. phenol red method in removal cell culture waste liquid according to claim 1, which is characterized in that in step (2) to
The buffer or ultrapure water of 3 times of volumes are added in cell culture waste liquid after ultrafiltration concentration.
3. phenol red method in removal cell culture waste liquid according to claim 1 or 2, which is characterized in that step (2) is slow
Fliud flushing is neutral buffered liquid.
4. phenol red method in removal cell culture waste liquid according to claim 1, which is characterized in that institute in step (3)
The volumetric concentration of the Triton X-114 of addition is 5%.
5. phenol red method in removal cell culture waste liquid according to claim 1, which is characterized in that 4 in step (3)
DEG C heat preservation 60min.
6. phenol red method in removal cell culture waste liquid according to claim 1, which is characterized in that will in step (4)
Step (3) gains, which are placed in 37 DEG C, to be continued to keep the temperature 60min.
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