CN101735984B - A Simple Method for Inoculating Cells with Tissue Engineering Scaffold Materials - Google Patents

Abstract

The invention provides a simple and effective method for realizing three-dimensional uniform inoculation of seed cells on a porous bioengineering support material. It is characterized in that a sterilized syringe commonly used in clinical practice is used as an inoculation tool, the required biological support material is put in from the back of the syringe, and then the cell suspension of the corresponding number of cells to be inoculated is inhaled. A certain amount of air is reserved in the syringe, the syringe port is closed, the syringe is withdrawn, and the negative pressure is used to make the cell suspension penetrate into the gap of the porous material, so as to realize the initial uniform distribution of seed cells in the scaffold material. Compared with the method of achieving initial three-dimensional uniform inoculation of scaffold materials in the existing tissue engineering practice, the method of the present invention does not need to spend a lot of money to purchase special instruments, is simple and easy to implement, and can obtain the initial three-dimensional uniform inoculation effect similar to that of special equipment. And because the time of negative pressure is short and the pressure is small, it will not damage the inoculated cells.

Description

A Simple Method for Inoculating Cells with Tissue Engineering Scaffold Materials

technical field

The invention relates to a method for realizing initial two-dimensional uniform cell inoculation on a porous support material in in vitro research and clinical application of tissue engineering.

Background technique

In the practice of tissue engineering, some cells need to be cultured and expanded in a two-dimensional environment in vitro to reach a certain number, or to achieve directional differentiation of cells through intervention measures during the culture process, and then seeded on the scaffold material; Some are seeded directly onto the scaffold material after harvesting the seed cells. The cell seeding process plays a very important role in the initial two-dimensional spatial distribution of cells and the subsequent tissue engineering culture differentiation process. To achieve uniform distribution of cells in the scaffold material at the initial stage of seeding is one of the important evaluation indexes of cell seeding method.

Currently used inoculation methods include static inoculation and dynamic inoculation. Among them, the static inoculation is to directly drop the cell suspension on the scaffold material, and allow the cell suspension to enter the interior of the scaffold material through osmosis. This method is currently a commonly used inoculation method due to its simple operation, but it cannot achieve uniform distribution of cells on the three-dimensional structure. At present, the more common dynamic inoculation methods include bioreactor method, centrifugation method, vacuum machine method, rotary bottle method, etc. These dynamic inoculation methods can achieve the uniform distribution of initial cells in the three-dimensional space of porous material scaffolds. However, these methods require the cooperation of specialized and expensive instruments, and the operation is relatively complicated. Therefore, there is an urgent need in this field to develop a new simple and easy cell seeding method to realize uniform distribution of cells in three-dimensional space at the initial stage of cell seeding.

Contents of the invention

The invention aims to use the equipment and devices commonly used in laboratories and clinics to simply and efficiently complete the initial uniform inoculation of seed cells on biological support materials. And in the process of cell inoculation, the shortest intervention time and minimum intervention intensity are adopted to maintain the biological activity of cells to the greatest extent.

In order to achieve the above object, the present invention provides a simple and efficient method, the steps are as follows:

a. Prepare the scaffold material for cell inoculation, and select the corresponding commonly used clinical disposable syringe specifications according to the size of the scaffold material.

b. Put the prepared stent material into the back of the syringe, put back the plunger of the syringe, and push out the excess air.

c. Inhale the cell suspension from the opening of the syringe, the volume of the cell suspension should just submerge the scaffold material. The cell concentration of the cell suspension varies according to different inoculation purposes.

d. Push out excess air, leaving only 1ml of air in the syringe except for the scaffold cell suspension complex.

e. Block the syringe port, withdraw the piston rod, increase the air in the syringe barrel to 3ml, and maintain this state for 5 seconds. Repeat the withdrawal in this way for two times.

f. Pull out the piston rod, pour out the excess cell suspension, take out the scaffold material and place it in a petri dish. At 37°C, in a cell culture incubator containing 5% CO2 for 2 hours, add the required medium after the cells are completely attached. Or it can be directly filled into the surgical defect after inoculation.

Description of drawings

Figure 1 Conventional static cell inoculation method. Cell suspension was dropped on the TCP scaffold material with a porosity of 60% for two hours, and the cell distribution was observed by MTT staining. It can be seen that active cells are mainly accumulated on the side of the material where the cell suspension is added. (MTT can act on the respiratory chain in the mitochondria of living cells, and form blue crystals of formazan under the action of succinate dehydrogenase and cytochrome C. Make active cells visible to the naked eye.)

Figure 2 shows that cells are inoculated on the same TCP scaffold material by the method of the present invention, and the distribution of cells is observed by MTT staining two hours later. It can be seen that the cells are evenly distributed in the material.

Fig. 3. Scanning electron micrographs of the central position after static inoculation of TCP scaffold material for 2 weeks, no cells survived. (×200)

Fig. 4 is a scanning electron micrograph of the central position after inoculation of the TCP scaffold material of the present invention for 2 weeks, showing cell survival. (×200)

Detailed ways

Example 1:

Human bone marrow stromal stem cells were seeded on TCP scaffolds for osteogenesis induction.

The bone marrow tissue collected clinically in the hospital was diluted with PBS solution, placed on the surface of an equal amount of Percoll liquid, and centrifuged at 3000rpm for 30 minutes. The milky white turbid mononuclear cell layer was collected for culture and expansion. After the cells were cultured to the second generation, the cell suspension was adjusted to 5×10 ⁶ /ml, and the scaffolds were prepared to be inoculated.

Prepare the TCP scaffold material with a size of 5mm×5mm×4mm, put the three scaffold materials into a 10ml syringe after being sterilized and dried. Aspirate 1ml of cell suspension, and reserve 1ml of air inside the syringe.

Block the opening of the syringe, withdraw the piston rod, so that the air inside the syringe reaches 3ml, and maintain for 5 seconds to slowly restore the pressure. Repeat this three times. Recover the excess cell suspension and continue to use it next time, move the inoculated cell-scaffold complex into a culture dish, let it stand in a cell culture incubator for 2 hours, and then add an osteogenic induction medium.

Example 2:

Human bone marrow stromal stem cells were seeded on TCP scaffolds for chondrogenic induction.

The bone marrow tissue collected in the hospital was diluted with PBS, placed on the surface of an equal amount of Percoll liquid, and centrifuged at 3000rpm for 30 minutes. The milky white turbid mononuclear cell layer was collected for culture and expansion. After the cells were cultured to the second generation, the cell suspension was adjusted to 14×10 ⁶ /ml, and the scaffolds were prepared to be inoculated.

Prepare the TCP scaffold material with a size of 5mm×5mm×4mm, put the three scaffold materials into a 10ml syringe after being sterilized and dried. Aspirate 1ml of cell suspension, and reserve 1ml of air inside the syringe.

Block the opening of the syringe, withdraw the piston rod, so that the air inside the syringe reaches 3ml, and maintain for 5 seconds to slowly restore the pressure. Repeat this three times. Recover the excess cell suspension and continue to use it next time, move the inoculated cell-scaffold complex into a culture dish, let it stand in a cell culture incubator for 2 hours, and then add a chondrogenic induction medium.

Example 3:

MC3T3-E1 cells were seeded on PDLLA scaffolds for osteogenesis induction experiments.

Cultivate MC3T3-E1 cell line in vitro, adjust the cell suspension to 5×10 ⁶ /ml after reaching a sufficient number, and prepare to inoculate scaffolds.

The PDLLA scaffold material was prepared with a size of 10mm×10mm×5mm, and three scaffold materials were put into a 10ml syringe after being sterilized and dried. Aspirate 2ml of cell suspension, and reserve 1ml of air inside the syringe.

Block the opening of the syringe, withdraw the piston rod, so that the air inside the syringe reaches 3ml, and maintain for 5 seconds to slowly restore the pressure. Repeat this twice. Recover the excess cell suspension and continue to use it next time, move the inoculated cell-scaffold complex into a culture dish, let it stand in a cell culture incubator for 2 hours, and then add an osteogenic induction medium.

Example 4:

Human osteoblasts were seeded on TCP scaffolds, and the interaction between osteoblasts and TCP scaffolds was studied.

Human cancellous bone collected clinically in the hospital, human osteoblasts were collected by digestion method, and cultured in a two-dimensional state in vitro. Collect the 3rd to 5th passage cells, adjust the cell suspension to 5×10 ⁶ /ml, and prepare to inoculate the scaffold.

Prepare the TCP scaffold material with a size of 5mm×5mm×4mm, put the three scaffold materials into a 10ml syringe after being sterilized and dried. Aspirate 1ml of cell suspension, and reserve 1ml of air inside the syringe.

Block the opening of the syringe, withdraw the piston rod, so that the air inside the syringe reaches 3ml, and maintain for 5 seconds to slowly restore the pressure. Repeat this three times. Recover the excess cell suspension and continue to use it next time, move the inoculated cell-scaffold complex into a culture dish, let it stand in a cell culture incubator for 2 hours, and then add an osteogenic induction medium.

Example 5:

Pig chondrocytes were seeded on PDLLA scaffolds for three-dimensional culture, and the effect of mechanical stress on three-dimensional cultured chondrocytes in vitro was observed.

The surface cartilage of the knee joint of 6-month-old pigs was collected, and pig chondrocytes were collected by digestion method. In order to prevent the degeneration of chondrocytes in vitro, the second-generation chondrocytes were collected, the cell suspension was adjusted to 14×10 ⁶ /ml, and the scaffolds were prepared.

The PDLLA scaffold material was prepared with a size of 10mm×10mm×5mm, and three scaffold materials were put into a 10ml syringe after being sterilized and dried. Aspirate 2ml of cell suspension, and reserve 1ml of air inside the syringe.

Block the opening of the syringe, withdraw the piston rod, so that the air inside the syringe reaches 3ml, and maintain for 5 seconds to slowly restore the pressure. Repeat this twice. Recover the excess cell suspension and continue to use it next time, move the inoculated cell-scaffold complex into a culture dish, let it stand in a cell culture incubator for 2 hours, and then add a chondrogenic induction medium containing TGF-β for culture. Subsequent low-frequency ultrasound stimulation was applied according to the experimental design.

Claims (2)

1. A method for biological material to realize initial three-dimensional uniform seeding of cells, its steps comprising: 1) Separating and culturing enough cells to be inoculated in the early stage, and adjusting the appropriate concentration of the cell suspension according to the purpose of inoculation, and preparing the scaffold material for inoculating the cells, and sterilizing it for use; 2) Select the corresponding inoculation tool according to the size of the scaffold material: the specification of the sterile syringe; 3) After pulling out the plunger of the syringe, put the prepared support material from the back of the syringe, put it back into the plunger of the syringe, and push out the excess air; 4) Inhale the prepared cell suspension from the opening of the syringe, and withdraw the plunger at the same time. The amount of the cell suspension should just submerge the scaffold material, and push out excess air. In the syringe, only keep 1ml except for the scaffold cell suspension complex. Air; 5) Block the syringe port, withdraw the piston rod, increase the air in the syringe barrel to 3ml, and maintain this state for 5 seconds, and repeat the withdrawal for three times; 6) Pull out the piston rod, pour out the excess cell suspension, take out the scaffold material and place it in a culture dish, and let it stand in a cell culture incubator containing 5% _CO2 at 37°C for 2 hours, and add the cell culture medium after the cells are completely attached. Culture medium is required. 2. The method for initial three-dimensional uniform inoculation of cells with the biological material according to claim 1, characterized in that: the sterile syringe is a clinically used disposable syringe or a sterile glass syringe.

Images