A method for constructing an artificial cell model in vitro based on xeno cell extracts

Abstract

This invention relates to the field of cell biology, and more particularly to a method for constructing an artificial cell model in vitro based on extracts from Xenopus laevis egg cells. The method includes: mixing cytoplasmic extracts from Xenopus laevis egg cells, demembranous sperm, and tubulin, dispersing the mixture in an oil phase to obtain a reconstructed system; reacting the reconstructed system at 20-24°C for at least 30 minutes, resulting in the formation of a phospholipid bilayer-based cytoplasmic membrane structure around the cell. This invention provides a method for constructing artificial cells by selecting cytoplasmic extracts from Xenopus laevis egg cells, adding specific substances, and reacting under specific conditions to allow the extracts to self-assemble into cell structures in vitro, simultaneously forming a cytoplasmic membrane. The method provided by this invention offers important clues to the origin and evolution of cells, and the resulting artificial cell structures are highly similar to normal cells, possessing significant application value.

Description

Technical Field

[0001] This invention relates to the field of cell biology technology, and in particular to a method for constructing an artificial cell model in vitro based on extracts from African Xenopus oocytes. Background Technology

[0002] Synthetic biology, a cutting-edge interdisciplinary field integrating knowledge from biology, chemistry, engineering, and other disciplines, primarily uses a bottom-up approach to assemble systems with life-like characteristics from non-living materials. Against this backdrop, how to simulate or synthesize artificial cells in vitro has become one of the core frontier issues in the field of synthetic biology for exploring the essence of life, constructing novel bioreactors, and developing intelligent drug delivery systems.

[0003] Artificial cells generally refer to microvesicle systems with cell membrane-like structures that can encapsulate biomolecules and perform specific biochemical reactions. Currently, the materials used to construct artificial cells mainly include phospholipids (liposomes), block copolymers (polymer vesicles), and proteins or peptides. These microsystems have shown great potential in mimicking cell compartmentalization, studying cellular metabolic pathways, and serving as artificial organelles for medical treatment.

[0004] Despite the promising prospects of artificial cell research, existing preparation technologies still face many challenges in practical applications, especially in terms of preparation efficiency and product quality.

[0005] First, while traditional preparation methods (such as thin-film hydration and reverse evaporation) are relatively simple to operate, they often rely on the self-assembly of molecules, a process with considerable randomness. This results in highly non-uniform product size distribution (high polydispersity) and poor batch-to-batch reproducibility, making it difficult to meet the standardization requirements of precision biological experiments or clinical applications.

[0006] Secondly, low encapsulation efficiency is a common problem faced by existing technologies. When constructing artificial cells containing complex enzyme systems or macromolecular genetic material, conventional methods struggle to ensure that the contents are efficiently and quantitatively encapsulated into the vesicles, resulting in a waste of expensive biological reagents and a low concentration of functional artificial cells.

[0007] Furthermore, while some emerging technologies (such as microfluidics or emulsion templates) have improved size control to some extent, they are often accompanied by complex preparation processes and high equipment costs. At the same time, if the organic solvents used in emulsion methods are not completely removed, the residual solvents may damage the stability of the vesicle membrane and even lead to the inactivation of internal bioactive substances, thereby affecting the biocompatibility and long-term stability of artificial cells. Summary of the Invention

[0008] To address the problems existing in the prior art, this invention provides a method for constructing an artificial cell model in vitro based on extracts from African Xenopus oocytes.

[0009] In a first aspect, the present invention provides a method for constructing artificial cells, comprising: A reconstructed system was obtained by dispersing cytoplasmic extracts, demembranous sperm, and tubulin from Xenopus laevis egg cells in an oil phase. The reconstructed system was reacted at 20-24°C for more than 30 minutes, and a cell membrane structure with a phospholipid bilayer was generated around it to encapsulate it.

[0010] This invention, through its research, has for the first time discovered that a reconstructed system based on cytoplasmic extracts from Xenopus laevis egg cells, with the addition of specific components, gradually evolves into a structure resembling that of normal cells after reacting under specific conditions. Furthermore, a phospholipid bilayer cytoplasmic membrane structure forms on the periphery (observed using fluorescence and electron microscopy), resulting in an artificial cell model with a structure similar to normal cells. This invention hypothesizes that the possible mechanism involves the abundant membrane components in the extract, the physical drive provided by the water-oil interface, the active transport of the cytoskeleton, and the combined effect of energy molecules, forming a spontaneously assembled, closed phospholipid bilayer membrane that encapsulates spontaneously assembled organelle structures, ultimately forming a cellular structure.

[0011] Furthermore, the reaction time of 30 minutes or more is defined as 30-600 minutes.

[0012] Furthermore, the cytoplasmic extract of the Xenopus laevis egg cells was obtained by the following method: the Xenopus laevis egg cells were centrifuged at 8000~12000g for 10~20 minutes, and the cytoplasmic fraction was collected; Preferably, the Xenopus laevis egg cells are centrifuged at 9000-11000g for 12-18 minutes.

[0013] Furthermore, the demembranous sperm is prepared by the following method: After obtaining the sperm of the African clawed frog, add lysophosphatidylcholine and treat at room temperature for 5-10 minutes; Preferably, the concentration of the lysophosphatidylcholine is 150~200 μg / mL.

[0014] Furthermore, the tubulin carries a signal reporter molecule; Preferably, the signal reporting molecule includes one or more of the following: small organic molecule fluorescent groups, near-infrared and silicon-based rhodamine probes, fluorescent proteins, photoactivated molecules, quantum dots, or gold nanoparticles.

[0015] The organic small molecule fluorescent groups described in this invention include: xanthracene derivatives (such as FITC, Rhodamine series, Oregon Green), cyanine derivatives (such as Cy3, Cy5, Sulfo-Cyanine), coumarin derivatives, AlexaFluor series or Atto dye series, etc.

[0016] The near-infrared and silicon-based rhodamine probes described in this invention include: silicon-based rhodamine (SiR) and its derivatives, carbon-based rhodamine, etc.

[0017] The fluorescent proteins described in this invention include: the green fluorescent protein family (e.g., GFP, EGFP), the red / orange fluorescent protein family (e.g., mCherry, TagRFP, DsRed, mOrange), and the cyan / yellow fluorescent protein family (e.g., CFP, YFP).

[0018] The photoactivated molecules described in this invention include: photoactivated GFP, mEos, or caged dyes (such as Caged Fluorescein), etc.

[0019] Furthermore, the reconstructed system is a dispersed liquid droplet.

[0020] Furthermore, the reconstructed system is reacted at 21~23°C for more than 45 minutes.

[0021] Furthermore, the cytoplasmic extract of the Xenopus laevis egg cells was in interphase or metaphase.

[0022] Furthermore, the reconstructed system also includes one or more of the following: cell membrane dyes, lipid dyes, DNA dyes, or organelle dyes (e.g., mitochondrial, Golgi dyes, etc.). Secondly, the present invention provides an artificial cell constructed by the aforementioned method.

[0023] Thirdly, the present invention provides a kit comprising the aforementioned artificial cells.

[0024] Fourthly, the present invention provides the application of the aforementioned artificial cells or the aforementioned kits in the preparation of intelligent drug delivery carriers, the development of clinical and environmental biosensors, or the biocatalytic synthesis in microbioreactors.

[0025] The artificial cells provided by this invention possess structures similar to normal cells (especially membrane structures), controllable material exchange properties, and compartmentalization effects of the internal environment: 1. It can be used to prepare intelligent drug delivery carriers. For example, it can serve as a highly biocompatible intelligent carrier for encapsulating small molecule drugs, nucleic acid drugs (such as DNA, mRNA, siRNA), or protein drugs. The selectively permeable membrane structure of artificial cells can effectively encapsulate drug molecules in the internal chamber, thereby protecting the drug from clearance by the host immune system or degradation by enzymes, and significantly prolonging the circulating half-life of the drug in vivo. By functionalizing the surface of artificial cells (e.g., by conjugating antibodies, aptamers, or ligands), they can be endowed with active targeting capabilities, enabling them to specifically recognize and accumulate in tumor tissue or specific lesion sites. In addition, the membrane structure or internal matrix of the artificial cells can be designed to respond to specific physiological microenvironmental stimuli (such as pH changes, specific enzyme concentrations, redox potentials, or temperatures). When the artificial cells reach the target site, environmental stimuli induce changes in membrane permeability or rupture, thereby achieving spatiotemporally controlled drug release and reducing toxic side effects on normal tissues.

[0026] 2. Artificial cells can be used for the development of clinical and environmental biosensors. For example, they can serve as miniaturized sensing units, utilizing the selective permeability of their membranes and internally encapsulated signal amplification systems to achieve ultrasensitive detection of clinical biomarkers or environmental pollutants. Specific biorecognition elements (such as enzymes, fluorescent probes, or gene circuits) can also be encapsulated within artificial cells. When specific biomarkers in blood or body fluids (such as glucose, uric acid, viral nucleic acids, or tumor exosomes) diffuse through the membrane into the cell, they trigger an internal cascade reaction, generating detectable optical or electrochemical signals. This confined space reaction can significantly increase the local substrate concentration, thereby amplifying the signal and improving detection sensitivity. Furthermore, artificial cells can be designed as environmentally responsive sensors to heavy metal ions, antibiotic residues, or organophosphorus pesticides in the environment. Because the artificial cell membrane has a natural barrier effect against external interfering substances, it can effectively reduce the interference of complex environmental matrices (such as sewage and soil leachate) on the detection results, improving the specificity and stability of on-site detection.

[0027] 3. It can be used in microbioreactors for biocatalytic synthesis, providing an independent, compartmentalized microreaction environment for constructing highly efficient in vitro multi-enzyme catalytic systems. Multiple enzymes involved in metabolic pathways can be co-encapsulated within artificial cells, transforming them into microbioreactors. This spatial proximity effect shortens the diffusion distance of intermediates, achieving substrate channeling, thereby significantly improving the overall rate and yield of multi-step enzymatic reactions. The internal environment of the artificial cell mimics the crowded environment of natural cells, helping to maintain enzyme conformational stability and preventing enzyme inactivation or aggregation during industrial catalysis. Furthermore, the selective permeability of the artificial cell membrane allows small substrate molecules to enter and exit products while retaining large enzyme molecules inside. This not only enables catalyst recovery and reuse but also simplifies subsequent product separation and purification steps, making it particularly suitable for the green synthesis of chiral pharmaceutical intermediates or high-value-added fine chemicals.

[0028] The present invention has the following beneficial effects: This invention selects extracts from Xenopus laevis egg cells and, by adding specific substances and reacting under specific conditions, enables them to self-assemble in vitro into cellular structures (including a nucleus, spindle apparatus, cytoskeleton, and structures similar to the endoplasmic reticulum), and simultaneously self-assemble into a cell membrane. The artificial cell structures provided by this invention are complete and similar to normal cell structures, possessing significant application value. Attached Figure Description

[0029] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0030] Figure 1 This is a flowchart of the extract droplet preparation and fluorescence imaging experiment provided in Example 1 of the present invention.

[0031] Figure 2 This is a diagram of the in vitro reconstruction of primitive interphase artificial cells from extract droplets provided in Embodiment 1 of the present invention.

[0032] Figure 3 This is a diagram of the in vitro reconstruction of primitive mid-stage artificial cells from extract droplets provided in Embodiment 1 of the present invention.

[0033] Figure 4 This is a schematic diagram of the experimental process for preparing extract droplets for transmission electron microscopy, provided in Embodiment 1 of the present invention.

[0034] Figure 5 This is an electron microscope ultrathin section of Xenopus oocyte extract droplet provided in Embodiment 1 of the present invention.

[0035] Figure 6 This is a schematic diagram of the experimental process for preparing extract droplets for scanning electron microscopy, provided in Embodiment 1 of the present invention.

[0036] Figure 7 This is a scanning electron microscope image of a droplet of Xenopus oocyte extract provided in Embodiment 1 of the present invention. Detailed Implementation

[0037] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0038] Unless otherwise specified, the experimental methods involved in the following embodiments are conventional methods in the art. For example, you can refer to the experimental manual in the art or follow the conditions recommended in the manufacturer's instructions.

[0039] Unless otherwise specified, all experimental materials and reagents used in the following examples are commercially available.

[0040] Example 1: Method for reconstructing primitive artificial cells in vitro using extracts from Xenopus laevis egg cells I. Experimental Materials 1. Xenopus laevis oocyte extract (interphase): Human chorionic gonadotropin (hCG) is injected into sexually mature female Xenopus laevis. High-quality oocytes are collected after ovulation. In preparing the interphase extract, cytochalasin B (CB), which disrupts the cytoskeleton, and cycloheximide (CH), which inhibits protein translation, are not added. The resulting extract ideally exhibits cell cycle characteristics, starting in interphase and progressing to the next cell cycle over time. This means that the cell cycle extract can exhibit different phases over time, rebuilding the nucleus in some periods and the spindle apparatus in others.

[0041] 2. African Xenopus oocyte extract (mid-stage): Human gonadotropin (hCG) was injected into sexually mature female African Xenopus. After ovulation, high-quality oocytes were collected and centrifuged at 10,000g for 15 minutes in a test tube. The cytoplasm was collected after centrifugation, specifically from the transparent upper part of the centrifuge tube (e.g., the upper part of the tube). Figure 1After centrifugation, the cells rupture, and different components are located in different positions in the centrifuge tube according to their different densities. The top layer is yellow lipids, the bottom layer is cell debris and some organelles, and the middle and upper part is brown and transparent, which is the cytoplasm. This part is collected.

[0042] 3. Demembranous sperm: (1) One day in advance, inject 100 units of human human chorionic gonadotropin into sexually mature male African clawed frogs. On the second day, take testis tissue from both sides of the frog and place it in pre-cooled XN buffer (15 mM NaCl, 60 mM KCl, 15 mM HEPES, 250 mM Sucrose, 0.5 mM Spermidine, 0.2 mM Spermine, 1 mM DTT, pH7.5).

[0043] (2) After washing with pre-cooled XN buffer, transfer the tissue to a glass homogenizer. Add 1 mL of XN buffer to the homogenizer and homogenize the testicular tissue on ice to extract the sperm. Collect the white suspension into a glass tube. Add 1 mL of XN buffer to the remaining testicular tissue in the homogenizer and wash 3 times. Collect all the liquid and centrifuge at 100 g horizontally at 4℃ for 1 min. Transfer the supernatant to another glass tube.

[0044] (3) Transfer the supernatant to another glass tube, centrifuge at 1500 g, 4℃ for 5 min, and a white precipitate of sperm will be visible at the bottom. Use a pipette to blow up the white part of the precipitate, collect the suspension, centrifuge at 1500 g, 4℃ for 5 min, repeat twice, and after each collection of the suspension, detect and remove red blood cells. After the last centrifugation, resuspend in a glass tube with 500 μL XN buffer, add an equal volume of 2× lysophosphatidylcholine (concentration of 330 μg / mL), mix with a pipette, and remove the membrane at room temperature for seven minutes.

[0045] (4) After the reaction is complete, terminate the reaction with three volumes of ice-cold XN buffer containing 3% BSA. Centrifuge at 1500 g, 4℃ for 5 min, collect the precipitate, and wash twice with XN buffer containing 3% BSA. Since the sperm are relatively loose after removing the membrane structure, be careful to slowly aspirate the supernatant with a pipette. Finally, suspend the precipitate in 1 mL of XN buffer containing 3% BSA, count the sperm under a microscope, and dilute to 1×10⁻⁶. 5 After collecting one microliter of sperm, the samples were aliquoted into PCR tubes, capped, and frozen in liquid nitrogen.

[0046] II. Experimental Methods 1. Constructing artificial cells (1) Take 20 μL of freshly prepared interphase Xenopus oocyte extract or interphase extract, and add it to a concentration of 1×10 5 Add 0.2 μL of demembranous sperm per μL, along with rhodamine-labeled or other color-labeled tubulin, and then add cell membrane dye (CellMask plasma membrane stain), lipid dye 3,3'-dihexyloxacarbocyanine (DHCC), and DNA fluorescent dye DAPI at a ratio of 1 / 1000. Alternatively, add mitochondrial dye Mitotracker or dyes for organelles such as the Golgi apparatus (for easier subsequent observation). Mix the above mixture thoroughly.

[0047] (2) Beforehand, attach a layer of fully transparent, inert material, fluorinated ethylene propylene (FEP) tape cut into a circle to the bottom of the dynamic dish to prevent the substances in the extract droplets from adsorbing onto the bottom of the dish and affecting the reconstruction effect. Then add 2 mL of dimethyl silicone oil with a viscosity of 500 to the dish.

[0048] (3) Slowly disperse 1 μL of the mixed extract into the silicone oil while simultaneously shaking the nozzle rapidly to disperse the extract into small droplets as it exits the nozzle. For larger droplets, pick them up with the nozzle and repeat the shaking and dispensing process until 1 μL of the mixed extract is dispersed into many very small extract droplets in the silicone oil (e.g., ...). Figure 1 (As shown).

[0049] (4) Place the dynamic dish containing the extract droplets on a fluorescence microscope beforehand to reconstruct the cells, so that imaging can be performed at different time points. Keep the room temperature at around 22°C and react at room temperature for more than 30 minutes. Some organelle-level life activities can then appear in some of the extract droplets.

[0050] This process can be visualized using fluorescence microscopy. For the interphase extract experimental group, the focus is on identifying droplets containing reconstructed round nuclei and large tubuloastromes labeled with tubulin. For the interphase extract preparation experimental group, the focus is on identifying droplets containing reconstructed spindle fibers. After identifying typical organelles such as nuclei or spindle fibers, the plasma membrane labeled with plasma membrane dyes and other fluorescence signals, such as membrane components labeled with lipid dyes (DHCC), are simultaneously imaged from the extract droplets.

[0051] 2. Imaging observation (1) In the small droplets formed by the interphase extract, the present invention can observe the following: Figure 2 The results shown are as follows: In image A, a droplet of extract from an interphase Xenopus oocyte, captured by confocal microscopy, displays typical interphase cell structures, including a cytoskeleton-like structure formed by purple tubulin (indicated by the arrow), and a membrane-like structure encasing the droplet (indicated by the arrow) as shown by green DHCC membrane dye. Red mitochondrial dye indicates the presence of mitochondria in the droplet, while blue DAPI dye shows the cell nucleus.

[0052] In B, the droplets of the extract from A are tilted at a 45° angle, and the resulting droplets exhibit a bowl-shaped structure.

[0053] In C, one layer of the three-dimensional structure of the extract droplet is shown, revealing structures such as the cell nucleus and plasma membrane.

[0054] In D, magnified images of the cell nucleus from C show the complete circular nuclear membrane (indicated by the arrow) and other membrane components in contact with the nuclear membrane, with DAPI showing the round nuclear structure formed after decondensation of sperm chromosomes.

[0055] Figure E shows extract droplets labeled with different colored dyes in other experimental groups, including a ring-shaped plasma membrane wrapped around the entire droplet labeled with purple dye, a cytoskeleton-like structure shown by tubulin labeled with red rhodamine, membrane components labeled with green membrane dye DHCC, and cell nuclei shown by DAPI.

[0056] In F, E is tilted at 60° to show the interface diagram of the extract droplet in contact with the inert culture dish. The purple plasma membrane dye and the green DHCC membrane dye both show the plasma membrane encapsulating the droplet (indicated by the arrow), as well as a network membrane structure similar to the endoplasmic reticulum (indicated by the arrow).

[0057] In the small droplets formed by the intermediate extract, the present invention can observe, as follows: Figure 3 The results shown are as follows: In image A, a confocal microscope image shows the spindle assembled in vitro from a mid-stage extract of Xenopus oocytes. Red rhodamine-labeled tubulin shows the typical spindle structure. Plasma membrane dyes and green DHCC membrane dyes show the membrane components clustered around the spindle. DAPI dyes show that demembranous sperm cells form chromosome-like structures arranged at the equatorial plate.

[0058] In Figure B, the extract droplet from mid-stage Xenopus oocytes exhibited a structure similar to mid-stage cells in vitro. This extract droplet contained a spindle and an internal network of membrane structures. The right image shows a cross-sectional view of one layer within this droplet; the purple plasma membrane dye reveals a ring of plasma membrane structures surrounding the droplet, as well as the network of membrane structures (indicated by the arrow). Simultaneously, the green DHCC membrane dye also shows a similar plasma membrane structure (indicated by the arrow). The magnified image shows the spindle structure and the membrane components clustered around the spindle, as well as the demembranous sperm located in the center of the spindle.

[0059] In step C, the cell cycle extract assembles into a structure similar to that of metaphase cells. The added demembranous sperm forms a chromosome-like structure, arranged at the equatorial plate. A green fluorescent probe of the Golgi apparatus reveals that the Golgi apparatus is also distributed within the extract droplets. Compared to the spindle formed in metaphase, this spindle formed from the cell cycle extract is more loosely spaced at the spindle poles and has a smaller diameter.

[0060] (2) To observe the ultrastructure of the reconstructed extract droplets using an electron microscope, especially the plasma membrane structure formed by the interphase extract, the extract droplet reconstruction should be performed first using the method mentioned above, without adding fluorescent dye. A mixture of fresh interphase extracts containing demembranous sperm and tubulin can be directly dispersed in silicone oil using a pipette tip and reconstructed in vitro at 22°C for 1 to 2 hours for subsequent sample preparation. Alternatively, the samples observed previously using fluorescence can be directly used for subsequent sample preparation.

[0061] (3) Prepare a 15 mL round-bottom centrifuge tube, add 3 mL of electron microscopy fixative (2.5% glutaraldehyde dissolved in 0.1 M dimethyl arsenate) to the bottom, and then transfer all the silicone oil mixed with the extract droplets into the centrifuge tube containing the fixative.

[0062] (4) Centrifuge at 5000 rpm and 4°C for 15 minutes using a horizontal centrifuge (Sigma centrifuge, model 3K15, rotor number 11133) (corresponding to a gravitational acceleration of 4480 g). If the speed is too low, the extract droplets will remain in the silicone oil and will not be able to centrifuge into the fixative at the bottom; if the speed is too high, the extract droplets will easily merge together at the bottom of the tube. Therefore, the speed and centrifugation time can be adjusted according to the retention of the droplets after centrifugation, so that the extract droplets can enter the fixative at the bottom of the tube without merging together at the bottom of the tube. The extract droplets enter the fixative solution at the bottom through centrifugation, and the outer periphery of the droplets is quickly fixed. Do not process the sample after centrifugation, and let the extract droplets continue to be fixed in the fixative overnight to ensure that the entire droplet is completely fixed.

[0063] (5) On the second day, remove the silicone oil from the top of the centrifuge tube and remove any excess fixative, or cut the centrifuge tube with a blade, leaving only a small portion containing fixative at the bottom (approximately 1 to 2 cm from the bottom). This will result in less residual silicone oil, less impact on the fixed droplets at the bottom during processing, and easier removal of the fixed extract droplets from the bottom. Wash the sample at least three times with 0.1 M dimethyl arsenate solution to remove the fixative and any remaining silicone oil on the surface.

[0064] (6) Remove the residual liquid and immediately add 1.5% (by mass / volume) of preheated and melted low-melting-point agarose (dissolved in 0.1 M dimethyl arsenate solution) to cover the bottom extract droplets. Place the centrifuge tube on ice to accelerate the solidification of the agarose. After about half an hour, remove the solidified agarose and place it in 0.1 M dimethyl arsenate solution. Under a stereomicroscope, use a blade to cut the solidified agarose into small cubes of about 1 cubic millimeter. Select the cubes containing the extract droplets and place them in a new 2.5% glutaraldehyde fixative for another hour. Then wash these fixed cubes three times with 0.1 M dimethyl arsenate solution for 10 minutes each time to remove the glutaraldehyde fixative (see experimental procedure). Figure 4 ).

[0065] (7) After cleaning, use a fixative prepared with 1% osmium tetroxide and 0.8% potassium ferrocyanide (dissolved in deionized water) to post-fix the sample for 1 hour. Place the sample in a fume hood and then rinse with deionized water at least 3 times for 5 minutes each time. Soak the cleaned sample in 1% uranium acetate solution at 4°C overnight, and then wash with deionized water 3 times for 5 minutes each time.

[0066] (8) Then dehydrate with a gradient of acetone concentrations: 30%, 50%, 70%, 90%, 100%, with two replacements of anhydrous acetone for 8 to 10 minutes each time. Then replace with a 3:1 mixture of anhydrous acetone and resin Epon-812 (anhydrous acetone: resin volume) for 1 to 3 hours, then replace with a 1:1 mixture (anhydrous acetone: resin volume) for 3 hours or overnight, then replace with a 1:3 mixture (anhydrous acetone: resin volume) for 2 hours or overnight, and then replace the mixture with pure resin. After two replacements with pure resin, polymerize at 65°C for 24 hours.

[0067] (9) Black extract droplets embedded in the polymerized resin can be seen. When cutting the sample, retain the black extract droplets to be cut. After the sample is properly cut, use an ultramicrotome to make ultrathin sections with a thickness of 70 μm. Use uranium acetate and lead citrate to stain the copper mesh with the ultrathin sections. Then observe and image under a transmission electron microscope FEI Tecnai G2 Spirit with an imaging voltage of 120 KV.

[0068] Transmission electron microscope section image as shown Figure 5 As shown, the left side is an electron microscope ultrathin section of a complete extract droplet. The extract droplet is well preserved, and its boundaries are clearly visible. The upper right half 1' is a magnified view of the area within the white box 1 on the left, the lower right half 2' is a magnified view of the area within the white box 2 in image 1', and the upper right 3' is a magnified view of the area within the white box 3. It can be seen that the boundary around the extract droplet is similar to the structure of the cell membrane. Under high-power electron microscopy, this membrane structure can also be seen to be composed of parallel phospholipid bilayers (indicated by the arrows).

[0069] (10) To observe the surface structure of the extract droplets, a scanning electron microscope (SEM) sample was prepared. A round-bottomed plexiglass column with a flat top was placed in a 15 mL round-bottomed centrifuge tube as a pad. A silicon wafer soaked in polylysine was placed on the column. The silicon wafer was about 9 mm × 9 mm in size. Then, 2 mL of 2.5% glutaraldehyde (prepared in 0.1 M dimethyl arsenate solution) was added to the centrifuge tube as an SEM fixative to ensure that the silicon wafer was immersed in the fixative.

[0070] (11) Transfer the previously reconstructed mixture of extract droplets and silicone oil to the centrifuge tube and centrifuge at 5000 rpm for 15 minutes in a horizontal centrifuge. After centrifugation, the extract droplets will be thrown onto the silicon wafer. Transfer the silicon wafer with the extract droplets to a small dish containing fresh 2.5% glutaraldehyde fixative and fix it again for 30 minutes. Then, wash the silicon wafer three times with 0.1 M dimethyl arsenate for 10 minutes each time (see the experimental procedure). Figure 6 ).

[0071] (12) Fix the silicon wafer with 1% osmium tetroxide and 0.8% potassium ferrocyanide in a fume hood for 1 hour, rinse with deionized water 3 times for 5 min each time, immerse the silicon wafer in 1% uranium acetate solution at 4°C overnight, rinse with deionized water 3 times for 5 min each time. Dehydrate the sample using an ethanol gradient: 30%, 50%, 70%, 90%, 100%, with two replacements of anhydrous ethanol for 5 min each time.

[0072] (13) The silicon wafer was dried and dehydrated using a Leica EM CP300 carbon dioxide critical point dryer, and then gold was sputtered using a Hitachi E-1045 ion sputtering instrument with the parameters set at 15 mA and 45 s. The thickness of the gold coating on the sample surface was about 4 to 5 nm. The sample was then observed under a Hitachi S4800 scanning electron microscope with an imaging voltage of 1.5 KV.

[0073] The results of scanning electron microscopy are as follows Figure 7 As shown: On the left is a scanning electron microscope image of a complete extract droplet. The spherical extract droplet is well preserved, and it can be seen that the extract droplet is encapsulated, but the contents inside are not visible. The right side 1' is a magnified view of a part of the white box 1 on the left, and the lower right half 2' is a magnified view of the part of the white box 2 in the image 1'. From the magnified view of 2', it can be seen that a small amount of circular film components are still attached to the surface of the spherical droplet (indicated by the arrow).

[0074] This shows that the surface of the Xenopus extract droplets is coated with substances, corresponding to the plasma membrane structure observed in the previous transmission electron microscopy, and there are also a small amount of membrane components adhering to the surrounding area.

[0075] In summary, this invention uses extracts of African Xenopus oocytes from different stages to reconstruct structures similar to interphase and metaphase cells in vitro. These structures are surrounded by structures similar to plasma membranes and contain typical organelles such as nuclei or spindles, as well as cytoskeleton and structures similar to endoplasmic reticulum.

[0076] The artificial cell preparation technology provided by this invention has broad application prospects, including basic research and translational applications: In basic scientific research, it may have the following application prospects: (1) Cell cycle research: This system allows for real-time, high-resolution observation of processes such as nuclear assembly, nuclear membrane breakdown and reconstruction, spindle assembly, and chromosome separation in vitro. Because this system is an open in vitro system, relevant substances, including but not limited to various dyes, small molecules, labeled proteins, and even other in vitro purified organelles, can be added to the extract beforehand to observe the changes and effects of these substances during the aforementioned processes. Compared to closed cell systems, the open system of this invention facilitates biochemical intervention.

[0077] (2) Membrane dynamics studies: The droplet system mentioned in this invention involves the assembly of a large number of membrane components, including the endoplasmic reticulum network, nuclear membrane, and cytoplasmic membrane. Therefore, this system is suitable for studying the formation, fusion, and transport mechanisms of cell membranes and nuclear membranes in vitro. Moreover, it is more open than cells, allowing for the addition of more exogenous components to study their effects on the membrane formation process.

[0078] (3) Organelle-related research: can be used to explore how organelles recognize, locate and communicate with each other.

[0079] (4) Virology research: Simulating the process of viral invasion, replication and bubbling.

[0080] In addition to the basic scientific research mentioned above, the artificial cells with nuclei and cell membranes prepared by this invention may also have the following specific applications in biology and medicine: (1) Applications of “cell factories” in synthetic biology, including protein synthesis, such as adding specific exogenous DNA to a reconstructed cell nucleus and utilizing the abundant transcription and translation machinery in the extract to make it a highly efficient protein production factory. Due to the encapsulation of the plasma membrane, the products can be protected to a certain extent, and the transport of substances can be controlled by modifying the membrane structure.

[0081] (2) Constructing other artificial cells: In the future, we can try to introduce in vitro purified chloroplasts, peroxisomes, etc. into the system to create artificial cells with new functions.

[0082] (3) Drug screening: Artificial cells carrying specific human disease genes (e.g., by adding modified DNA) can be constructed to screen drugs that target cell cycle, nuclear function or membrane-related processes.

[0083] (4) Toxicity test: The artificial cells prepared in this invention can be used as a platform for cellular environmental toxicity testing.

[0084] (5) Drug or gene delivery system: The artificial cell constructed in this invention is similar to a natural giant vesicle and can be modified, for example, to load drugs, proteins, mRNA, or gene editing tools such as CRISPR-Cas9. In addition, membrane proteins can be modified or targeted ligands, such as antibodies or peptides, can be added to enable them to specifically recognize and fuse with specific target cells (such as cancer cells). Furthermore, its membrane structure can be designed to respond to specific signals (such as low pH or specific enzymes) to achieve precise release.

[0085] (6) Cell mimicry therapy in medicine: For diseases with cell function defects, artificial cells with complementary functions (such as providing a missing enzyme or signaling molecule) can be designed to mimic the corresponding artificial cells to perform their functions.

[0086] (7) Personalized medical models in regenerative medicine: Artificial cells can be assembled using patient-derived DNA or key proteins to test personalized treatment plans.

[0087] In summary, this invention establishes a flexible and convenient in vitro artificial cell regeneration platform. Besides the previously mentioned use of biochemical methods, such as adding inhibitors or antibodies to neutralize relevant proteins to identify key factors in membrane formation and further study the mechanism, this open system can also incorporate more substances and even organelles to enhance its functions. Furthermore, optogenetic or small molecule control systems can be introduced to achieve precise spatiotemporal control of cell cycle transitions and membrane permeability. Additionally, its feasibility as a targeted delivery vector can be tested.

[0088] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for constructing artificial cells, characterized in that, include: A reconstructed system was obtained by dispersing cytoplasmic extracts, demembranous sperm, and tubulin from Xenopus laevis egg cells in an oil phase. The reconstructed system was reacted at 20-24°C for more than 30 minutes, and a cell membrane structure with a phospholipid bilayer was generated around it to encapsulate it.

2. The method according to claim 1, characterized in that, The cytoplasmic extract of the Xenopus laevis egg cells was obtained by the following method: the Xenopus laevis egg cells were centrifuged at 8000~12000g for 10~20 minutes and the cytoplasmic fraction was collected. Preferably, the Xenopus laevis egg cells are centrifuged at 9000-11000g for 12-18 minutes.

3. The method according to claim 1 or 2, characterized in that, The demembranous sperm was prepared by the following method: After obtaining the sperm of the African clawed frog, add lysophosphatidylcholine and treat at room temperature for 5-10 minutes; Preferably, the concentration of the lysophosphatidylcholine is 150~200 μg / mL.

4. The method according to any one of claims 1-3, characterized in that, The tubulin carries a signal reporter molecule; Preferably, the signal reporting molecule includes one or more of the following: small organic molecule fluorescent groups, near-infrared and silicon-based rhodamine probes, fluorescent proteins, photoactivated molecules, quantum dots, or gold nanoparticles.

5. The method according to any one of claims 1-4, characterized in that, The reconstructed system is a dispersed liquid droplet.

6. The method according to any one of claims 1-5, characterized in that, The reconstructed system was reacted at 21-23°C for at least 45 minutes.

7. The method according to any one of claims 1-6, characterized in that, The cytoplasmic extract of the Xenopus laevis egg cells was in the interphase or metaphase.

8. An artificial cell, characterized in that, It is constructed by the method described in any one of claims 1-7.

9. A reagent kit, characterized in that, Includes the artificial cell as described in claim 8.

10. The artificial cell of claim 8, or the kit of claim 9, is used in the preparation of intelligent drug delivery carriers, the development of clinical and environmental biosensors, or in biocatalytic synthesis in microbioreactors.

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