CN101038283B

CN101038283B - Method for maintaining stabilization of perfusate pH value, oxygen partial pressure and carbon dioxide partial pressure

Info

Publication number: CN101038283B
Application number: CN2007100176117A
Authority: CN
Inventors: 李延海; 韩太真; 谢雯
Original assignee: Xian Jiaotong University
Current assignee: Xian Jiaotong University
Priority date: 2007-04-03
Filing date: 2007-04-03
Publication date: 2011-04-06
Anticipated expiration: 2027-04-03
Also published as: CN101038283A

Abstract

In order to solve the problem of using a large amount of mixed gas in life science experiments, the experiment cost is high, and the pH value, Po ₂ and Pco ₂ of the perfusate are unstable, the present invention discloses a low-cost, maintainable pH value of the perfusate, Po _{2 and Pco 2} . The Pco ₂ stabilization method is to use a set of water to isolate the air to prevent the precipitation of dissolved gas in the perfusate that is directly exposed to the air. The mixed gas is introduced into the perfusate of the first container for a short period of time to saturate the mixed gas in the perfusate, and the air in the second container is replaced with the mixed gas to saturate the water in the third container. When the perfusate is pumped out by the peristaltic pump according to the experimental needs, as the perfusion liquid level in the first container drops, the mixed gas in the second container is sucked into the first container, and the water in the third container enters the second container at the same time, Therefore, the stability of the pH of the perfusate and the pressure Po ₂ and Pco ₂ of the mixed gas in the first container is ensured.

Description

A kind of stable method of perfusate pH value, partial pressure of oxygen and partial pressure of carbon dioxide of keeping

Technical field

The present invention relates to a kind ofly in Life Science Experiment, keep perfusate pH value, partial pressure of oxygen (Po ₂) and partial pressure of carbon dioxide (Pco ₂) stable method.

Background technology

The interior environment of biosome (is the physicochemical environment of extracellular fluid, comprises temperature, ion concentration, osmotic pressure, pH value, Po ₂And Pco ₂Deng) constant in relatively keeping tissue and the cell normal metabolic is necessary.Wherein pH constant relatively (fluctuation range 7.35～7.45 of human body pH) is that the regulatory function (handling acid and alkaline metabolic product) that relies on the interior various buffer systems of body and lung and kidney realizes.This processing acid-base material of body content with ratio, keep the relative constant process of pH value and be called acid base equilibrium (acid-base balance).The Po of body fluid ₂And Pco ₂The constant relatively eubolism for cell and tissue is essential.Life science by using artificial perfusate's method, is kept the normal physicochemical environment of extracellular fluid often to the research of isolated cells, tissue or organ, makes it near physiological status.All adopt both at home and abroad at present 95% oxygen and 5% carbon dioxide mix gas are fed in the perfusate, to keep its pH value, Po ₂With Pco ₂Stable.This is not only the prerequisite of tissue or organ survival, also is the key factor that influences the experimental result science.But present employed method is to continue ventilation in the container that opens wide, and is about to breather line and directly puts into perfusate.In case it is too small to stop ventilation or air-flow, the pH value that is directly exposed to airborne perfusate can increase gradually along with the prolongation of time, and partial pressure of oxygen can reduce gradually.The defective of the method is: (1) because functional experiment often needs to carry out continuously 1,2 hour even the longer time, combination gas needs round-the-clock supply, causes the experimental cost height; (2) because the instability of venting pressure and speed can cause perfusate pH value and Po ₂Fluctuation, the ventilation excessive velocities promptly can cause pH value reduction, Po ₂And Pco ₂Higher, ventilation speed is crossed and is then caused the rising of pH value, Po slowly ₂And Pco ₂On the low side, thus the illusion of result of study easily caused; (3) the amount difference of size, shape and the perfusate of different experiments (or experimenter) institute use container will be kept suitable perfusate pH value, Po ₂And Pco ₂Then need different aeration speeds, control suitable aeration speed usually and will depend on experience; (4) continue ventilation, easily cause pH value, the Po of different parts perfusate ₂And Pco ₂Unbalanced, on the low side as ventilation position pH value, and Po ₂And Pco ₂Higher.

Summary of the invention

Use a large amount of combination gass in order to solve in the Life Science Experiment, the experimental cost height of prior art, and perfusate pH value, Po ₂And Pco ₂Instability problem, the invention provides a kind of cheaply, can keep perfusate pH value, Po ₂And Pco ₂Stable method has the advantages that design is ingenious, make simply, be convenient to operate.

For reaching above purpose, the present invention takes following technical scheme to be achieved:

A kind of stable method of perfusate pH value, partial pressure of oxygen and partial pressure of carbon dioxide of keeping is characterized in that, comprises the steps:

(1) container of three equal volume of use, described container is divided into first container, second container and the 3rd container, is respectively charged into perfusate and water in first container and the 3rd container, and the volume of water is no less than the volume of perfusate; Second container is vacant; Three containers have two-tube sealing-plug with one respectively inserts in the container with the vessel port sealing and with two-tube, and wherein inserting the two-tube of first container and the 3rd container is long a, weak point, and long tube is inserted the bottom of solution, and short tube is inserted container top air chamber; Insert the two-tube short tube that is of second container, and be communicated with first seal of vessel short tube and the 3rd seal of vessel long tube beyond the Great Wall beyond the Great Wall, the 3rd seal of vessel short tube and air communication beyond the Great Wall respectively;

(2) first seal of vessel long tube is beyond the Great Wall connected a threeway, this threeway links to each other with combination gas with peristaltic pump again;

(3) opening threeway makes combination gas be communicated with the long tube of first container, and block simultaneously being communicated with of this long tube and peristaltic pump, led to combination gas at least 10 minutes for the perfusate in first container, make the saturated mixed gas of perfusate and replace the air in second container and then make the water saturation of the 3rd container, too much combination gas is discharged by the short tube of the 3rd container;

(4) open threeway and make peristaltic pump be communicated with the long tube of first container, and block simultaneously being communicated with of this long tube and combination gas, start peristaltic pump perfusate is flowed out by the required flow velocity of experiment.

In the such scheme, the preferably distilled water that the 3rd container pours in the described step (1); Two-tube first seal of vessel short tube and the 3rd seal of vessel long tube beyond the Great Wall beyond the Great Wall that be communicated with respectively in described second container preferably adopts silicone tube to be communicated with; In the described step (2), first seal of vessel long tube beyond the Great Wall preferably also adopts silicone tube to connect with being connected of threeway.

Compare with the existing method of perfusate continuous charge of giving, the present invention adopts the method for a cover water secluding air, has prevented from directly to be exposed to separating out of the interior dissolved gas of airborne perfusate, does not therefore need can stablize Po to the perfusate continuous charge ₂With the pH value.When the combination gas (10 minutes) that in the first container perfusate, feeds the short period, just can make the combination gas in the perfusate saturated, and be combination gas the air displacement in second container, and then the saturated again water in the 3rd container.When perfusate need be extracted out by peristaltic pump by experiment, decline along with perfusion liquid level in first container, combination gas in second container is sucked and enters first container, water in the 3rd container enters second container simultaneously, the 3rd vessel level descend again outside air is pumped in, thereby guaranteed mixture pressure and perfusate pH in first container, Po ₂And Pco ₂Stable.

The present invention removes above-mentioned perfusate pH value, the Po of keeping ₂And Pco ₂Outside the constant relatively advantage, also have and to save a large amount of combination gass, simple, the convenience operation of making.

Description of drawings

Fig. 1 is for realizing the device synoptic diagram of the inventive method.Among the figure: 1, threeway; 2, flexible pipe; 3, sealing-plug; 4, air chamber; 5, long tube; 6, short tube; 7, perfusate; 8, air; 9, pure water;

Fig. 2 is pH value, the Po of perfusate in 5 hours of the inventive method embodiment 1 ₂And Pco ₂Variation diagram.Wherein Fig. 2 a be the pH value over time; Fig. 2 b is Po ₂Over time; Fig. 2 c is Pco ₂Over time.

Fig. 3 directly is exposed to pH value, the Po of airborne perfusate in 5 hours for using ₂And Pco ₂Variation diagram.Wherein, Fig. 3 a be the pH value over time; Fig. 3 b is Po ₂Over time; Fig. 3 c is Pco ₂Over time.

Embodiment

The present invention is described in further detail below in conjunction with drawings and Examples.

Embodiment 1

As shown in Figure 1, a kind of perfusate pH value, Po of keeping ₂And Pco ₂Stable method comprises the steps:

(1) container of three equal volume of use as Clear glass bottles and jars, is respectively charged into perfusate 7 and distilled water 9 in container A and container C, and distilled water 9 volumes are not less than the volume of perfusate 7, and container B is vacant; Three containers have two-tube sealing-plug 3 with the vessel port sealing with one respectively, and insert in the container two-tube, and wherein inserting the two-tube of container A and C is a long tube 5, a short tube 6, and long tube 5 is inserted the bottom of solution, and short tube 6 is inserted container top air chamber 4; Long tube 5, short tube 6 can adopt stainless-steel tube or glass tube etc.Insert the two-tube of container B and be short tube 6, and use short tube 6 on the silicone tube connection container A sealing-plug 3 and the long tube 5 on the container C sealing-plug 3 respectively, the short tube 6 on the container C sealing-plug 3 is communicated with outside air.

(2) the 5 usefulness silicone tubes of the long tube on the container A sealing-plug 3 are connected a medical threeway 1, it links to each other with mixture pipe with a peristaltic pump 10, and peristaltic pump 10 is used for perfusate 7 is supplied with biological tissue and cell.

(3) switch of the medical threeway 1 of rotation makes the combination gas that contains 95% oxygen and 5% carbon dioxide gas by the long tube 5 among the silicone tube connection container A, gives the perfusate in first container 7 logical combination gass, and blocks simultaneously being communicated with of this long tube 5 and peristaltic pump 10; When continuing logical combination gas after 10 minutes, the air in the saturated successively perfusate 7 of combination gas, the container B also is replaced into combination gas, and then saturated distilled water 9 in the container C.

(4) switch of the medical threeway 1 of rotation makes the long tube 5 of peristaltic pump 10 connection container A, and blocks simultaneously being communicated with of this long tube 5 and combination gas, starts peristaltic pump 10 and makes perfusate 7 by the required flow velocity outflow of experiment; Decline along with 7 liquid levels of perfusate in the container A, combination gas in the container B is sucked and enters container A, distilled water in the container C enters container B simultaneously, in the decline of container C liquid level is pumped into outside air again, thereby guaranteed container A, container B and container C internal gas pressure basically identical, and then guaranteed perfusate 7pH value, Po ₂And Pco ₂Stable.The perfusate 7 that uses in the present embodiment is artificial cerebrospinal fluid (perfusion that is used for stripped brain tissue of Neuroscience Research and neurocyte).

Embodiment 2

The method step of present embodiment is substantially with embodiment 1, and institute's difference is that the perfusate 7 that container A is packed into is a physiological saline in step (1); The water that container C is packed into is pure water; In step (3), combination gas continues 12 minutes in the feeding container A physiological saline 7.

The inventive method is not limited to the perfusate of above embodiment, tests employed perfusate for other isolated cells, tissue and organ, all can be suitable for equally as locke solution, ringer solution, tyrode's solution etc.

Fig. 2 shows the continuous recording pH value of perfusate, the Po of the embodiment of the invention 1 ₂And Pco ₂5 hours situation of change shows that from Fig. 2 a result the pH value changes very little, and remains in the physiological pH scope (7.4 ± 0.5); Fig. 2 b and Fig. 2 c show its Po in 5 hours respectively ₂And Pco ₂Substantially constant.Directly be exposed to airborne perfusate, its pH value, Po and use ₂And Pco ₂Situation of change is seen shown in Figure 3.Among Fig. 3 a, its pH value is along with the prolongation of time increases gradually, and when the time reached 5 hours, its pH value increased to more than 8; Po among Fig. 3 b, Fig. 3 c ₂And Pco ₂That reduces is also very obvious, away from the normal value level.Above experimental result has illustrated that the inventive method has solved life science in vitro tissue or organ research, and the stability problem that the artificial perfusate uses has significant technique effect to the true reappearance experimental result.

Claims

1. A method for maintaining the stability of perfusate pH value, oxygen partial pressure and carbon dioxide partial pressure, is characterized in that, comprises the steps:

(1) Use three containers of the same volume, the containers are divided into the first container, the second container and the third container, respectively fill the first container and the third container with perfusate and water, the volume of water is quite large The volume of the perfusate; the second container is empty; the three containers are respectively closed with a sealing plug with a double tube and the double tube is placed in the container, wherein the double tube of the first container and the third container is inserted One long and one short, the long tube is placed at the bottom of the solution, the short tube is placed in the upper air chamber of the container; the double tubes placed in the second container are both short tubes, and are respectively connected to the short tube on the sealing plug of the first container and the The long tube on the sealing plug of the third container, the short tube on the sealing plug of the third container communicates with the air;

(2) Connect the long pipe on the sealing plug of the first container to a tee, which is connected to the peristaltic pump and the gas mixture again;

(3) Open the tee to connect the mixed gas to the long tube of the first container, and at the same time block the connection between the long tube and the peristaltic pump, and pass the mixed gas to the perfusate in the first container for at least 10 minutes to saturate and mix the perfusate gas and replace the air in the second container, thereby saturating the water in the third container, and the excess gas mixture is discharged through the short pipe of the third container;

(4) Open the tee to connect the peristaltic pump to the long tube of the first container, and at the same time block the connection between the long tube and the mixed gas, start the peristaltic pump to make the perfusate flow out to supply living tissues and cells at the flow rate required for the experiment.

2. the method for maintaining perfusate pH value, oxygen partial pressure and carbon dioxide partial pressure stability according to claim 1, it is characterized in that, the water that the 3rd container fills in described step (1) is double distilled water.

3. The method for maintaining the perfusate pH value, partial pressure of oxygen and partial pressure of carbon dioxide according to claim 1 is characterized in that, the double tubes in the second container are respectively connected to the short pipes on the sealing plug of the first container. The connection between the tube and the long tube on the sealing plug of the third container is connected by a silicone tube; the connection between the long tube on the sealing plug of the first container and the tee is connected by a silicone tube.