CN-121972243-A - Microfluidic biological deep reservoir preparation method and blood cell concentration prediction method

CN121972243ACN 121972243 ACN121972243 ACN 121972243ACN-121972243-A

Abstract

The invention provides a preparation method of a microfluidic biological deep reservoir and a blood cell concentration prediction method, which comprises the steps of manufacturing a microfluidic biological deep reservoir structure by adopting a PDMS (polydimethylsiloxane) material based on a preset microfluidic chip die, wherein the microfluidic biological deep reservoir structure comprises a plurality of sub-treatment reservoirs which are connected in a directional manner, each sub-treatment reservoir is the same in size, a plurality of directional connection channels are arranged between the sub-treatment reservoirs which are connected in a directional manner, each sub-treatment reservoir corresponds to one reservoir of the deep reservoir, extracting primary nerve cells from pregnant ICR mice, implanting the primary nerve cells into the sub-treatment reservoirs of the microfluidic biological deep reservoir structure, culturing the inoculated microfluidic biological deep reservoir structure in an incubator, and infecting neurons by jointly applying CheRiff and jRCaMP b viruses in the culture process so that the neurons express the optogenetic proteins and calcium ion indicators at the same time, and finally culturing to obtain the microfluidic biological deep reservoir.

Inventors

GUI LILI
WANG KE
HAN ZHUO
Dai Wanqi
DENG YIN
XU KUN

Assignees

北京邮电大学

Dates

Publication Date: 20260505
Application Date: 20260116

Claims (10)

1. A method for preparing a microfluidic biological deep reservoir, the method comprising the steps of: Based on a preset microfluidic chip die, a microfluidic biological depth storage pool structure is manufactured by adopting a PDMS material, the microfluidic biological depth storage pool structure comprises a plurality of sub-treatment pools which are connected in a directional mode, the sizes of the sub-treatment pools are the same, a plurality of directional connection channels are arranged between the sub-treatment pools which are connected in a directional mode, the directional connection channels are provided with arc blind area structures, and each sub-treatment pool corresponds to one storage pool of the depth storage pool; Extracting fetal mouse cortex tissue from pregnant ICR mice, dissociating primary nerve cells from the fetal mouse cortex tissue, and implanting the primary nerve cells into a sub-treatment pool of a microfluidic biological deep reservoir structure; Culturing the inoculated micro-fluidic biological deep reserve pool structure in an incubator, and infecting neurons by jointly applying CheRiff and jRCaMP b viruses in the culture process so that the neurons express the optogenetic protein and the calcium ion indicator at the same time, and finally culturing to obtain the micro-fluidic biological deep reserve pool.
2. The method of preparing a microfluidic biological depth reservoir according to claim 1, wherein the arc-shaped dead zone structure is provided with a plurality of sub-arc-shaped dead zones extending in an arc shape in a direction opposite to a directional extension direction of the directional connection channel.
3. The method for preparing the microfluidic biological depth reservoir according to claim 2, wherein the radius of the sub arc-shaped dead zone is R, R is more than or equal to 0.015mm and less than or equal to 0.025mm, and the angle range of the sub arc-shaped dead zone is delta, and delta is more than or equal to 250 degrees and less than or equal to 290 degrees.
4. The method for preparing a microfluidic biological depth reservoir according to claim 1 or 2, wherein the directional connection channel is further provided with a channel port structure, the channel port structure is provided with a plurality of symmetrically arranged right triangle structures, and the pointing direction of the right triangle structures is the same as the directional extension direction of the directional connection channel.
5. A method for predicting blood cell concentration, the method comprising the steps of: Collecting and analyzing a blood sample to obtain a blood cell concentration value of the blood sample, performing simulation calculation based on the blood cell concentration value to obtain blood cell concentration values at a plurality of time points, constructing a blood cell concentration sequence, and encoding into a three-channel sequence based on the blood cell concentration sequence; Determining optical stimulation signals of a first channel, a second channel and a third channel based on the three channel sequence, and applying optical stimulation to a first sub-processing pool of a plurality of sub-processing pools directionally connected in the microfluidic biological depth storage pool according to any one of claims 1-4 based on the optical stimulation signals; The method comprises the steps of collecting a light stimulus response signal of a sub-processing pool in a microfluidic biological depth storage pool by adopting image collecting equipment, inputting the light stimulus response signal into a prediction module, wherein the prediction module is provided with an output layer, and outputting a blood cell concentration value prediction result of a prediction time position through the output layer.
6. The method according to claim 5, wherein in the step of obtaining the blood cell concentration values at a plurality of time points by performing a simulation calculation based on the blood cell concentration values and constructing the blood cell concentration sequence, the blood cell concentration value at the next time point is simulated based on the blood cell concentration value at the first time point by using the blood cell concentration value currently measured as the blood cell concentration value at the first time point, the blood cell concentration values at a plurality of time points are calculated by using the same simulation calculation method, and finally the blood cell concentration values at all time points are constructed as the blood cell concentration sequence.
7. The blood cell concentration predicting method according to claim 6, wherein in the step of calculating the blood cell concentration values at a plurality of time points by the same analog calculation method, the blood cell concentration variation is calculated by using the following formula, and the blood cell concentration value at the next time point is determined based on the raw blood cell concentration value and the blood cell concentration variation; Wherein, the Blood cell concentration values representing the current time point, Represents the amount of change in blood cell concentration at the current time point, Representing a predetermined delay parameter when In the time-course of which the first and second contact surfaces, Is a preset constant value when When the calculated history value is used.
8. The method according to claim 5, wherein in the step of encoding the three-channel sequence based on the blood cell concentration sequence, the blood cell concentration variation range is determined based on the blood cell concentration value and the variation threshold value at the previous time point in the blood cell concentration sequence, wherein in the blood cell concentration sequence, if the blood cell concentration value at the current time point is greater than the maximum value of the blood cell concentration variation range, the first channel encoding is 1, if the blood cell concentration value at the current time point is smaller than the minimum value of the blood cell concentration variation range, the second channel encoding is 1, if the blood cell concentration value at the current time point is within the blood cell concentration variation range, the first channel and the second channel both encoding are 0, and the third channel employs rate encoding, the blood cell concentration value at the current time point is compared with the accumulation threshold value, if the blood cell concentration value at the current time point is greater than the accumulation threshold value is 1, if the blood cell concentration value at the current time point is not greater than the accumulation threshold value, the accumulation threshold value is still further compared with the accumulation threshold value at the next time point, and the accumulation threshold value is still not greater than the accumulation threshold value.
9. The method according to claim 5, wherein in the step of determining optical stimulation signals of the first channel, the second channel, and the third channel based on the three channel sequence, applying optical stimulation to a first sub-processing cell of the plurality of sub-processing cells directionally connected in the microfluidic biological depth reservoir based on the optical stimulation signals, the first channel, the second channel, and the third channel are respectively provided with stimulation positions in the first sub-processing cell of the microfluidic biological depth reservoir, and applying optical stimulation to the first sub-processing cell of the microfluidic biological depth reservoir based on the encoding results of the first channel, the second channel, and the third channel.
10. The method according to claim 5, wherein in the step of collecting optical stimulus response signals of sub-processing pools in the microfluidic biological depth pool by using the image collection device, inputting the optical stimulus response signals into the prediction module, collecting a preset number of response images from each sub-processing pool, superposing the response images collected from each sub-processing pool to obtain a superposed image, screening the target neuron position of each sub-processing pool based on the superposed image, collecting pixel values of the target neuron position for each response image of each sub-processing pool, constructing an input vector, and combining the input vectors of a plurality of sub-processing pools to obtain the optical stimulus response signals.

Description

Microfluidic biological deep reservoir preparation method and blood cell concentration prediction method Technical Field The invention relates to the technical field of biological neural networks, in particular to a preparation method of a microfluidic biological deep reservoir and a blood cell concentration prediction method. Background In recent years, with the development of emerging interdisciplines such as biomedical engineering, materials science and the like, the microfluidic technology has a rich research value and a wide application prospect in the fields of biosensors, clinical detection, organ chip construction and the like, and particularly has advantages in long-term culture of in-vitro biological neural networks. The microfluidic technology refers to a technology for processing or controlling fluid in a micro-nano level space by using a specially designed micro-channel, and a microfluidic chip constructed by using the microfluidic technology is mainly manufactured by using Polydimethylsiloxane (PDMS for short), and the transparent material and good biocompatibility of the microfluidic chip provide favorable conditions for culture survival and optical observation of nerve cells. Pool calculation is a low training cost Recurrent Neural Network (RNN) with wide application in timing information processing. The increase of the number of directional connection layers in the deep reservoir through the interior of the reservoir is more effective in realizing layered information processing capability, richer reservoir state number, larger memory capacity and more complex dynamic characteristics. The biological neural network for in-vitro culture realizes modularized and directional neural cell culture by a microfluidic technology, and a biological depth reservoir capable of carrying out signal stimulation and acquisition is further constructed. Compared with an artificial neural network, the biological deep reserve pool has the advantages of low energy consumption, strong generalization, good robustness, integration of storage and calculation and the like. In recent years, many biological neural network studies based on in vitro culture have demonstrated excellent characteristics of biological reservoirs in accomplishing some typical tasks, such as stimulation by optogenetic means at university of northeast japan, performing voice-digital classification tasks using multi-module biological reservoirs cultured in vitro, which exhibit good generalization ability. Similarly, the university of indiana in the united states uses brain organoids cultured in vitro to perform tasks such as voice recognition, and the plasticity of biological neural networks is studied while achieving higher accuracy. These studies have all verified the feasibility of biological reservoirs for information processing, but there is still a limitation that the layers of the biological reservoirs of the prior art are all commonly connected, and common connection results in the bi-directional growth of nerves between the two layers, and the prior art does not simulate the complex connection characteristics in the brain through directional connection, so the data processing accuracy of the final biological reservoir is low. Disclosure of Invention In view of the foregoing, embodiments of the present invention provide a method of preparing a microfluidic biological deep reservoir that obviates or mitigates one or more of the disadvantages of the prior art. In one aspect, the invention provides a method for preparing a microfluidic biological deep reservoir, comprising the steps of: Based on a preset microfluidic chip die, a microfluidic biological depth storage pool structure is manufactured by adopting a PDMS material, the microfluidic biological depth storage pool structure comprises a plurality of sub-treatment pools which are connected in a directional mode, the sizes of the sub-treatment pools are the same, a plurality of directional connection channels are arranged between the sub-treatment pools which are connected in a directional mode, the directional connection channels are provided with arc blind area structures, and each sub-treatment pool corresponds to one storage pool of the depth storage pool; Extracting fetal mouse cortex tissue from pregnant ICR mice, dissociating primary nerve cells from the fetal mouse cortex tissue, and implanting the primary nerve cells into a sub-treatment pool of a microfluidic biological deep reservoir structure; Culturing the inoculated micro-fluidic biological deep reserve pool structure in an incubator, and infecting neurons by jointly applying CheRiff and jRCaMP b viruses in the culture process so that the neurons express the optogenetic protein and the calcium ion indicator at the same time, and finally culturing to obtain the micro-fluidic biological deep reserve pool. With the above-described approach, since the axons of neurons tend to grow along the edges of the structures when they enco