Microfluidic chip for separating platelet-rich plasma, and method for using same

Abstract

Disclosed in the present invention are a microfluidic chip for separating platelet-rich plasma (PRP) and a method for using same. The microfluidic chip comprises a sample injection chamber, a red blood cell storage chamber, a red blood cell collection chamber, a PRP collection chamber, a PPP collection chamber, an interface layer detection channel, a red blood cell extraction channel, a PRP extraction channel, and a PPP extraction channel. The three collection chambers, i.e., the red blood cell collection chamber, the PRP collection chamber, and the PPP collection chamber, are each provided with a corresponding negative pressure output channel and negative pressure port. By means of the microfluidic chip of the present invention, PRP can be separated in a sealed chip throughout the whole process, thereby avoiding external contamination. The operations such as centrifugation and layering, red blood cell extraction, plasma extraction, and PRP enrichment in a PRP preparation process are all performed continuously, thereby effectively shortening the time required for PRP preparation.

Description

A microfluidic chip for separating platelet-rich plasma and its application method Technical Field

[0001] This invention relates to the field of blood separation, and specifically discloses a microfluidic chip for separating platelet-rich plasma and its usage method. Background Technology

[0002] Platelet-rich plasma (PRP) is a platelet concentrate obtained by centrifuging autologous whole blood, with a platelet concentration at least twice the baseline concentration. Since PRP preparation currently mostly uses autologous whole blood, PRP usually refers to autologous PRP, which mainly contains platelets, fibrin, and leukocytes. Alpha granules in platelets release a large number of growth factors upon platelet activation. The proportions of these growth factors correspond to the physiological state in the body, playing a crucial role in accelerating wound healing, promoting tissue regeneration and repair, and relieving pain. In recent years, it has been widely applied in various medical fields, showing broad application potential and development prospects.

[0003] Methods for preparing PRP mainly include single-stage centrifugation, double-stage centrifugation, and triple-stage centrifugation, with double-stage centrifugation being the most widely used. Existing PRP preparation kits and equipment require multiple transfers of blood cells (red blood cells and plasma) and extraction of the PRP product during the preparation process, making it cumbersome. Furthermore, these extractions are all performed manually, leading to product contamination and excessively long preparation times. Secondly, because most centrifugation uses tubular or cup-shaped containers, the boundary layer between plasma and red blood cells is thin after blood centrifugation, making it difficult to determine and control the boundary layer's position. This results in unstable PRP enrichment folds, low PRP collection volumes, and high levels of residual red blood cells. Summary of the Invention

[0004] To solve the aforementioned technical problems, the inventor, based on his extensive experience in the field of blood separation, developed a microfluidic chip for separating platelet-rich plasma and its usage method.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: a microfluidic chip for separating platelet-rich plasma, comprising an injection chamber, a red blood cell storage chamber, a red blood cell collection chamber, a PRP collection chamber, a PPP collection chamber, a boundary layer detection channel, a red blood cell extraction channel, a PRP extraction channel, and a PPP extraction channel, wherein the three collection chambers of the red blood cell collection chamber, the PRP collection chamber, and the PPP collection chamber are each equipped with a corresponding negative pressure output channel and a negative pressure interface.

[0006] Furthermore, a boundary layer detection channel is connected below the sample injection chamber, and a red blood cell storage chamber is connected below the boundary layer detection channel; during centrifugation, the negative pressure output module controls the boundary layer between red blood cells and plasma to enter the detection channel by transferring red blood cells.

[0007] Furthermore, a red blood cell extraction channel is connected to the left side of the red blood cell storage chamber, and a red blood cell collection chamber is connected to the top of the red blood cell extraction channel.

[0008] The red blood cell storage chamber is mainly used to store the lower layer of red blood cells after centrifugation and stratification of blood. The volume of the red blood cell storage chamber of the chip is required to be lower than the minimum red blood cell volume ratio of the minimum injection blood volume, so as to ensure that the boundary layer is always in the injection chamber after centrifugation and stratification within the injection volume range.

[0009] The red blood cell collection chamber is used to collect the lower layer of red blood cells after blood centrifugation; the volume of the chip red blood cell collection chamber is required to be higher than the highest red blood cell volume ratio of the maximum blood sample volume, so as to ensure that the red blood cells extracted within the sample volume will not completely fill the chamber.

[0010] The red blood cell extraction channel connects the bottom of the red blood cell storage chamber to the top of the red blood cell collection chamber. When negative pressure is applied to the red blood cell collection chamber, red blood cells from the red blood cell storage chamber can be extracted and transferred into the red blood cell collection chamber. The boundary layer after centrifugation is then controlled to enter the interface detection channel.

[0011] Furthermore, the bottom of the PRP extraction channel is connected to the middle of the boundary layer detection channel, and the top of the PRP extraction channel is connected to the PRP collection chamber.

[0012] The PRP extraction channel is used to connect the middle part of the boundary layer detection channel to the PRP collection chamber. When negative pressure is output to the PRP collection chamber, the white film layer and plasma in the detection channel and its sample injection chamber can be extracted and sent into the PRP collection chamber for centrifugation.

[0013] Furthermore, the bottom of the PPP extraction channel is connected to the PRP collection chamber, and the top of the PPP extraction channel is connected to the PPP collection chamber.

[0014] The PRP collection chamber is used to collect the upper plasma layer after centrifugation. After enrichment at a higher rotation speed, the lower plasma layer becomes the product PRP. The PPP collection chamber is used to collect the upper plasma layer after centrifugation and enrichment in the PRP chamber.

[0015] The PPP extraction channel is used to connect the PPP collection chamber and the PRP collection chamber. After centrifugation and enrichment, the bottom layer of the PRP collection chamber is the product PRP, and the top layer is PPP. By outputting negative pressure to the PPP collection chamber, the PPP in the upper layer of the PRP collection chamber can be extracted and entered into the PPP collection chamber.

[0016] Furthermore, the bottom of the negative pressure output channel is connected to the red blood cell collection chamber, the PRP collection chamber, and the PPP collection chamber, respectively, and the top of the negative pressure output channel is connected to the negative pressure interface. The negative pressure output channel is used to connect the negative pressure interface to each collection chamber, transmitting the pressure output from the negative pressure output source to each collection chamber to achieve the extraction of red blood cells, plasma, and PPP.

[0017] Furthermore, the negative pressure interface is used to connect the negative pressure source pipeline to the chip, ensuring that the output negative pressure can enter the chip. The negative pressure interface is mainly used to connect the negative pressure source pipeline to the chip, ensuring that the output negative pressure can enter the chip.

[0018] This invention also discloses a method for using a microfluidic chip for separating platelet-rich plasma, comprising the following steps: S1: After adding a whole blood sample to the sample inlet chamber, centrifugation begins. Under centrifugal force, the whole blood sample is centrifuged and separated into a plasma layer, a red blood cell layer, and a white blood cell layer as the boundary between the two; S2: At this time, the red blood cell storage chamber and the stratification detection channel are filled with red blood cells, and the plasma layer and the white blood cell layer are located in the sample inlet chamber; S3: Negative pressure is applied to the red blood cell collection chamber, and the red blood cells in the red blood cell storage chamber enter the red blood cell collection chamber through the red blood cell extraction channel; S4: As some red blood cells in the red blood cell storage chamber are transferred to the red blood cell collection chamber, the white blood cell layer and its plasma layer are separated. The pressure gradually decreases, and after a period of time, the white membrane layer enters the boundary layer detection channel. The negative pressure output of the red blood cell collection chamber is immediately stopped, and the interface movement stops. S5: Negative pressure is output to the PRP collection chamber, and the white membrane layer and its upper plasma layer enter the PRP collection chamber through the PRP extraction channel until all the upper plasma layer enters the PRP collection chamber, at which point the negative pressure output is stopped. S6: The centrifugation speed is increased to enrich the PRP in the plasma in the PRP collection chamber. S7: After enrichment, negative pressure is output to the PRP collection chamber, and the upper plasma layer in the PRP collection chamber enters the PRP collection chamber through the PRP extraction channel. Finally, the product PRP is obtained in the PRP collection chamber.

[0019] Furthermore, during the extraction process in step S7, the white blood cell content in the PRP can be adjusted by precisely controlling the extraction amount of the white membrane layer.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] 1. Using this microfluidic chip, platelet-rich plasma can be separated entirely within a closed chip, avoiding external contamination.

[0022] 2. The centrifugation, red blood cell extraction, plasma extraction, and PRP enrichment processes in PRP preparation are all performed continuously, which can effectively shorten the PRP preparation time.

[0023] 3. The microchannels on the microfluidic chip ensure accurate determination of the position of the white membrane layer during the separation and extraction process, reducing platelet loss and increasing the enrichment factor of the product PRP, while significantly reducing the residual amount of red blood cells and white blood cells.

[0024] 4. The enrichment factor of the final product PRP is controllable. By changing the volume of the final product PRP, PRPs with different enrichment factors can be obtained.

[0025] 5. With the help of matching instruments, PRP preparation can be fully automated. After anticoagulated whole blood enters the microfluidic chip, the equipment automatically completes the preparation operation and outputs the product PRP, reducing manual operation, lowering the risk of contamination, and improving the efficiency of PRP preparation. Attached Figure Description

[0026] Figure 1 is a schematic diagram of the overall structure of the microfluidic chip of the present invention.

[0027] Figure 2 is a schematic diagram of the microfluidic chip chamber and its channel connection according to the present invention.

[0028] Figure 3 is a schematic diagram of the gas path and interface of the microfluidic chip of the present invention.

[0029] Figure 4 is a schematic diagram of the overall structure of the microfluidic chip of the present invention.

[0030] Figure labels: 1. Sample injection chamber; 2. Boundary layer detection channel; 3. Red blood cell storage chamber; 4. Red blood cell collection chamber; 5. Red blood cell extraction channel; 6. PRP collection chamber; 7. PRP extraction channel; 8. PPP collection chamber; 9. PPP extraction channel; 10. Negative pressure interface; 11. Negative pressure output channel. Detailed Implementation

[0031] Preferred embodiments of the present disclosure will be described in detail below with reference to the accompanying drawings. Before description, it should be understood that the terminology used in the specification and appended claims should not be construed as limited to its general and dictionary meaning, but rather should be interpreted based on the principle of allowing the inventors to appropriately define the terminology for the best interpretation, and based on its meaning and concept corresponding to the technical level of the present invention. Therefore, the description herein is merely a preferred example for illustrative purposes and is not intended to limit the scope of the invention; thus, it should be understood that other equivalent implementations and modifications can be made without departing from the spirit and scope of the invention.

[0032] Referring to Figures 1-4, this embodiment provides a microfluidic chip for separating platelet-rich plasma (PRP), including an injection chamber 1, a red blood cell storage chamber 3, a red blood cell collection chamber 4, a PRP collection chamber 6, a PPP collection chamber 8, a boundary layer detection channel 2, a red blood cell extraction channel 5, a PRP extraction channel 7, and a PPP extraction channel 9. Each of the three collection chambers (red blood cell collection chamber 4, PRP collection chamber 6, and PPP collection chamber 8) is equipped with a corresponding negative pressure output channel 11 and a negative pressure interface 10. The injection chamber 1, boundary layer detection channel 2, red blood cell storage chamber 3, red blood cell collection chamber 4, PRP collection chamber 6, and PPP collection chamber 8 are located at the bottom layer (first layer) of the chip, as shown in Figure 2; the red blood cell extraction channel 5, PRP extraction channel 7, and PPP extraction channel 9 are located at the second layer of the chip, and the corresponding gas paths and their interfaces are located at the third layer of the chip, as shown in Figure 3. Each chip layer is independent of the others.

[0033] In this embodiment, a boundary layer detection channel 2 is connected below the sample injection chamber 1, and a red blood cell storage chamber 3 is connected below the boundary layer detection channel 2; during centrifugation, the negative pressure output module controls the boundary layer between red blood cells and plasma to enter the detection channel.

[0034] In this embodiment, the red blood cell storage chamber 3 is connected to a red blood cell extraction channel 5 on its left side, and the red blood cell extraction channel 5 is connected to a red blood cell collection chamber 4 at its top.

[0035] In this embodiment, the bottom of the PRP extraction channel 7 is connected to the middle of the boundary layer detection channel 2, and the top of the PRP extraction channel 7 is connected to the PRP collection chamber 6.

[0036] In this embodiment, the bottom of the PPP extraction channel 9 is connected to the PRP collection chamber 6, and the top of the PPP extraction channel 9 is connected to the PPP collection chamber 8.

[0037] In this embodiment, the bottom of the negative pressure output channel 11 is connected to the PPP collection chamber 8, and the top of the negative pressure output channel 11 is connected to the negative pressure interface 10.

[0038] In this embodiment, the negative pressure interface 10 is used to connect the negative pressure source pipeline to the chip, ensuring that the output negative pressure can enter the chip.

[0039] This embodiment also discloses a method for using a microfluidic chip for separating platelet-rich plasma, including the following steps:

[0040] S1: After adding the whole blood sample into the sample injection chamber 1, start centrifugation. Under the action of centrifugal force, the whole blood sample is centrifuged and separated into plasma layer, red blood cell layer, and the boundary layer between the two is white membrane layer.

[0041] S2: At this time, both the red blood cell storage chamber 3 and the stratification detection channel 2 are filled with red blood cells, and the white membrane layer is located in the sample injection chamber 1. At this time, the volume of the red blood cell storage chamber of the chip is required to be lower than the minimum red blood cell volume ratio of the minimum sample blood volume. This is to ensure that the boundary layer is always in the sample injection chamber 1 after centrifugation and stratification within the sample injection volume range.

[0042] S3: When negative pressure is output to the red blood cell collection chamber 4, the red blood cells in the red blood cell storage chamber 3 enter the red blood cell collection chamber 4 through the red blood cell extraction channel 5. At this time, the volume of the chip red blood cell collection chamber is required to be higher than the highest red blood cell volume ratio of the maximum injection blood volume, so as to ensure that the red blood cells extracted within the injection volume will not completely fill the chamber.

[0043] S4: Due to the decrease in red blood cells in the red blood cell storage chamber 3, the white membrane layer and its plasma layer descend. After a period of time of red blood cell extraction, the white membrane layer enters the boundary layer detection channel 2, and the negative pressure output of the red blood cell collection chamber 4 is immediately stopped, and the interface movement stops;

[0044] S5: Negative pressure is output to PRP collection chamber 6, and the white membrane layer and its upper plasma layer enter PRP collection chamber 6 through PRP extraction channel 7 until all upper plasma layer enters PRP collection chamber 6, at which point the negative pressure output stops;

[0045] S6: Increase the centrifugation speed to enrich PRP in the PRP collection chamber 6;

[0046] S7: After enrichment, negative pressure is output to the PPP collection chamber 8, and the upper plasma in the PRP collection chamber 6 is introduced into the PPP collection chamber 8 through the PPP extraction channel 9. Finally, the product PRP is obtained in the PRP collection chamber 6.

[0047] In this embodiment, during the extraction process in step S7, the white blood cell content in PRP can be adjusted by controlling the amount of white membrane layer extracted.

[0048] The working principle of this invention will now be explained through more detailed experimental data.

[0049] Example 1: 5 mL of whole blood was added to sample chamber 1 and centrifuged. The centrifugation procedure was as follows: centrifugation at 3500 rpm for 300 s to separate red blood cells from plasma; red blood cells were extracted and entered into red blood cell collection chamber 4 within 300 s at 2450 rpm, and the boundary layer (white membrane layer) was controlled to enter the target position of the boundary layer detection channel 2. All plasma and its white membrane layer were extracted and entered into plasma collection chamber 7; the speed was increased to 3500 rpm and centrifuged for 420 s to enrich platelets in the plasma. The upper plasma layer, i.e., PPP, was extracted and entered into the corresponding chamber, and the lower plasma layer was the product PRP. The detection results of the components in each chamber after separation are shown in Table 1 below.

[0050]

[0051] Example 2: 5 mL of whole blood was added to sample injection chamber 1 and centrifuged. The centrifugation procedure was as follows: centrifugation at 3500 rpm for 300 s to separate red blood cells from plasma; red blood cells were extracted and entered into red blood cell collection chamber 4 at 2450 rpm for 300 s, and the boundary layer (white membrane layer) was controlled to enter the target position of the boundary layer detection channel 2, with only the upper plasma layer and a small portion of the white membrane layer being extracted and entered into the PRP collection chamber; the speed was increased to 3500 rpm and centrifuged for 420 s to enrich platelets in the plasma. The upper plasma layer, i.e., PPP, was extracted and entered into the corresponding chamber, while the lower plasma layer was the product PRP. The detection results of the components in each chamber after separation are shown in Table 2 below.

[0052]

[0053] Example 3: In this example, 15 mL of whole blood was added to the sample injection chamber for centrifugation. The centrifugation procedure was as follows: centrifugation at 3000 rpm for 300 s to separate red blood cells from plasma; red blood cells were extracted into the red blood cell collection chamber at 2450 rpm for 300 s, and the boundary layer (white membrane layer) was controlled to enter the target position of the boundary layer detection channel. All plasma and a small portion of the white membrane layer were extracted and entered into the plasma collection chamber; the speed was increased to 3500 rpm and centrifuged for 420 s to enrich platelets in the plasma. The upper plasma layer, i.e., PPP, was extracted and entered into the corresponding chamber, while the lower plasma layer was the product PRP. The detection results of the components in each chamber after separation are shown in Table 3 below.

[0054]

[0055] Comparative Example:

[0056] Compare this invention with any commercially available separation and extraction method:

[0057]

[0058] As can be seen from the above comparative examples, the separation and extraction method described in the invention can prepare a larger volume of PRP product at the same enrichment factor, and can achieve a higher platelet recovery rate. At the same time, there are fewer erythrocyte and leukocyte residues in the leukocyte-rich PRP product, and the leukocyte content in the leukocyte-rich PRP can be adjusted according to actual needs.

[0059] The foregoing has described in detail specific parts of the present invention. It is clear to those skilled in the art that these specific descriptions are merely preferred embodiments, and the scope of the present invention is not limited thereto. Therefore, the essential scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A microfluidic chip for separating platelet-rich plasma, characterized in that: The system includes a sample injection chamber, a red blood cell storage chamber, a red blood cell collection chamber, a PRP collection chamber, a PPP collection chamber, a boundary layer detection channel, a red blood cell extraction channel, a PRP extraction channel, and a PPP extraction channel. Each of the three collection chambers (red blood cell collection chamber, PRP collection chamber, and PPP collection chamber) is equipped with a corresponding negative pressure output channel and negative pressure interface. The boundary layer detection channel is connected to the lower part of the sample injection chamber, and the red blood cell storage chamber is connected to the lower part of the boundary layer detection channel. The red blood cell extraction channel is connected to one side of the red blood cell storage chamber, and the red blood cell collection chamber is connected to the top of the red blood cell extraction channel. The bottom of the PRP extraction channel is connected to the middle of the boundary layer detection channel, and the top of the PRP extraction channel is connected to the PRP collection chamber. The bottom of the PPP extraction channel is connected to the PRP collection chamber, and the top of the PPP extraction channel is connected to the PPP collection chamber.

2. The microfluidic chip for separating platelet-rich plasma according to claim 1, characterized in that: During centrifugation, the negative pressure output module controls the entry of the red blood cell and plasma boundary layer into the detection channel by transferring red blood cells.

3. A microfluidic chip for separating platelet-rich plasma according to claim 1, characterized in that: The red blood cell storage chamber is used to store the lower layer of red blood cells after blood centrifugation and separation, and the red blood cell collection chamber is used to collect the lower layer of red blood cells after blood centrifugation and separation.

4. A microfluidic chip for separating platelet-rich plasma according to claim 1, characterized in that: The PRP extraction channel is used to connect the middle part of the boundary layer detection channel with the PRP collection chamber. When negative pressure is output to the PRP collection chamber, the white film layer and plasma in the detection channel and its sample injection chamber can be extracted and put into the PRP collection chamber for centrifugation.

5. A microfluidic chip for separating platelet-rich plasma according to claim 1, characterized in that: The PRP collection chamber is used to collect the upper plasma layer after centrifugation, and the PPP collection chamber is used to collect the upper plasma layer enriched by centrifugation in the PRP chamber.

6. A microfluidic chip for separating platelet-rich plasma according to claim 1, characterized in that: The bottom of the negative pressure output channel is connected to the PPP collection chamber, and the top of the negative pressure output channel is connected to the negative pressure interface.

7. A microfluidic chip for separating platelet-rich plasma according to claim 1, characterized in that: The negative pressure interface is used to connect the negative pressure source pipeline to the chip, so that the output negative pressure can enter the chip.

8. A method of using the microfluidic chip according to any one of claims 1 to 7, characterized in that: The following steps are included: S1: After adding the whole blood sample into the injection chamber, centrifugation begins. Under the action of centrifugal force, the whole blood sample is centrifuged and separated into plasma layer, red blood cell layer, and the boundary layer between the two is the white membrane layer. S2: At this time, both the red blood cell storage chamber and the stratified detection channel are filled with red blood cells, and the white membrane layer is located in the sample injection chamber; S3: Negative pressure is output to the red blood cell collection chamber, and red blood cells in the red blood cell storage chamber enter the red blood cell collection chamber through the red blood cell extraction channel; S4: Due to the reduction of red blood cells in the red blood cell storage chamber, the white membrane layer and its plasma layer descend. After a period of time of red blood cell extraction, the white membrane layer enters the boundary layer detection channel, and the negative pressure output of the red blood cell collection chamber is immediately stopped, and the interface movement stops. S5: Output negative pressure to the PRP collection chamber, and the white membrane layer and its upper plasma layer enter the PRP collection chamber through the PRP extraction channel until all the upper plasma layer enters the PRP collection chamber, and then stop the negative pressure output; S6: Increase the centrifugation speed to enrich PRP in the PRP collection chamber; S7: After enrichment, negative pressure is applied to the PPP collection chamber to allow the upper plasma in the PRP collection chamber to enter the PPP collection chamber through the PPP extraction channel, and finally the product PRP is obtained in the PRP collection chamber.

9. The method of using the microfluidic chip according to claim 8: characterized in that: During the extraction process in step S7, the white blood cell content in PRP can be adjusted by controlling the amount of white membrane layer extracted.

Images