A cell preparation method based on reprogramming and directed evolution screening and application thereof

Abstract

The application discloses a preparation method of a cell preparation and belongs to the technical field of biological medicine and regenerative medicine. The preparation method is characterized by the following process logic: first, performing lineage resetting treatment on somatic cells, and then performing directional evolution screening on the obtained reset cells. Any intermediate operation such as freezing, recovery and expansion can be arranged between the two processes, and the somatic cells are not limited in species, autologous or allogeneic sources. The application solves the defects of simple directional evolution genomic damage accumulation and poor in-vivo stress tolerance of simple reset cells through specific process combination, and the two processes form a synergistic effect. The application also provides a cell preparation prepared by the method and application of the cell preparation in preparation of anti-aging drugs and skin aging improvement cosmetics.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and regenerative medicine technology, and relates to a cell preparation process, as well as the cell preparation obtained by the process and its application. Background Technology

[0002] Cell therapy has important applications in tissue repair and aging intervention, but existing mainstream technologies have insurmountable technical defects.

[0003] Individual directed evolution processes rely on continuous cell division and passage to complete the screening. The division process causes continuous shortening of cell telomeres and continuous accumulation of gene mutations. After long-term continuous passage, the instability of the cell genome increases significantly. The higher the screening intensity and the more passages, the higher the risk of cell genome abnormalities, making it difficult to meet the safety requirements for long-term in vivo application.

[0004] Individual lineage resetting technology can restore senescent somatic cells to a youthful state, repair telomeres, and reset methylation patterns. However, young cells that have not undergone stress acclimatization will rapidly lose activity after entering a stress environment and will not be able to maintain long-term function.

[0005] The order in which the processes are performed has a decisive impact on the final cell performance. If the primitive somatic cells are first subjected to directed evolutionary screening and then lineage reprogramming is carried out, the reprogramming process will remove the adaptive traits obtained from cell screening, and it will be impossible to improve the two types of technical defects mentioned above at the same time.

[0006] Currently, existing technologies do not disclose a complete process combination scheme that strictly limits the implementation of lineage reset followed by directed evolutionary screening, and lack a standardized preparation route that can simultaneously take into account both genomic safety and cellular environment tolerance.

[0007] Purpose of the invention

[0008] This invention provides a method for preparing cell preparations. By defining a fixed sequence of steps, it combines lineage resetting treatment with directed evolution screening, thereby simultaneously overcoming the technical defects of unstable genomes in directed evolution and weak stress resistance in cells with lineage resetting alone. The somatic cells used in this invention can be of human or mammalian origin, and can be autologous or allogeneic cells. The scope of application is not limited by species, autologous or allogeneic source.

[0009] Technical solution

[0010] A method for preparing a cell preparation includes the following steps:

[0011] (a) Somatic cells were subjected to lineage reset treatment to obtain reset cells;

[0012] (b) Using the reset cells to apply selection pressure for directed evolutionary screening;

[0013] Step (a) is performed before step (b). Step (b) can be performed directly after step (a) is completed. Alternatively, one or more intermediate operations such as cell cryopreservation, thawing, amplification, detection, and storage can be set between step (a) and step (b). After the intermediate operations, the cells are reset and still used as raw materials for step (b).

[0014] The implementation method of lineage resetting, the type of pressure to be selected, the indicators used for directed evolution screening, and whether to add gene editing or small molecule pretreatment are all optional optimization schemes that can be freely adjusted according to the actual application scenario. For specific optimization schemes, please refer to dependent claims 2 to 11.

[0015] Working principle and collaborative technology effects

[0016] Pre-lineage reset treatment restores somatic cells to the baseline state of young cells. The telomere loss and gene mutations generated by subsequent directed evolution and passage will not be superimposed on the aging damage inherent in the original cells, thus reducing the risk of cellular genome abnormalities from the source.

[0017] Post-selective evolutionary screening exerts selective pressure on rejuvenated cells, enabling them to autonomously develop environmental adaptation characteristics under continuous stress, thus significantly improving their survival ability under various stress conditions.

[0018] The sequence of processes is a necessary condition for achieving synergistic effects. The implementation method of prioritizing pedigree reset completely eliminates the stress resistance characteristics obtained by cell selection, and cannot simultaneously achieve the dual advantages of genomic stability and high environmental tolerance. Only by strictly following the process logic of first implementing pedigree reset and then implementing directed evolutionary selection can the two processes compensate for each other's inherent defects and produce synergistic effects that cannot be achieved by a single process.

[0019] Whether or not buffer operations such as storage, amplification, and detection are set between the two steps, the baseline of the youthful cells established by the previous lineage reset will not be changed. Regardless of whether there is an intermediate buffer, the core technical effect of this invention, which balances safety and cell viability, can be achieved.

[0020] The entire process of this invention can be completed using conventional cell culture, passage, and detection equipment, and it is suitable for large-scale industrial production of various autologous and allogeneic somatic cells from human and mammalian sources.

[0021] Beneficial effects

[0022] The core protection boundaries of this invention are clear, focusing only on the process combination logic of first resetting the lineage and then directing the evolution. The supporting process conditions can be flexibly replaced, making it difficult to simply circumvent the core process by fine-tuning the parameters.

[0023] This invention simultaneously addresses two major industry pain points: the accumulation of genome damage from isolated directed evolution and the poor stress tolerance of cells when simply resetting them, resulting in unexpected synergistic technological effects.

[0024] This invention is highly versatile, with no species or autologous / allogeneic source restrictions on somatic cells, and can be adapted to different stress screening conditions and different pretreatment processes.

[0025] The stress factor concentrations, screening rounds, and index judgment thresholds described in the embodiments are merely exemplary preferred parameters and do not constitute a limitation on the scope of protection of the present invention; the preferred critical screening survival rate of the present invention is 40%; in this embodiment, the telomere length threshold is set to 6.5kb, which is between the unsafe comparative example of 4.2kb and the experimental group of the present invention of 7.8kb, and is a safe critical value that can be reasonably selected by those skilled in the art.

[0026] Terminology Explanation

[0027] Lineage reset treatment: Various cell treatment methods that can restore aging somatic cells to a youthful state.

[0028] Directed evolutionary selection: The process of applying selection pressure in vitro and selecting dominant cell clones through single or multiple rounds of passage culture.

[0029] Selection stress: various stress conditions artificially applied to induce adaptive selection of cells.

[0030] Somatic cells: differentiated cells derived from multicellular organisms.

[0031] The above terminology definitions are for the purpose of assisting in understanding the present invention only and are not intended to limit the scope of protection of the claims. Detailed Implementation

[0032] The cell types, stress factor concentrations, screening rounds, and screening index thresholds described in the following embodiments are preferred exemplary parameters of the present invention and are not intended to limit the scope of protection. The somatic cells described in this invention are not limited to the types listed herein; any species, autologous, or allogeneic somatic cells can be adapted to this preparation process. Those skilled in the art can freely replace the reset methods, stress types, and screening indexes, and can select any species, autologous, or allogeneic somatic cells. Additional steps such as gene editing and small molecule pretreatment can also be added, as long as the core process logic of first implementing lineage reset and then implementing directed evolution screening is followed, all of which fall within the scope of protection of this invention.

[0033] Example 1: Preparation of human dermal fibroblast preparation

[0034] Abdominal skin tissue was collected from healthy volunteers, and human dermal fibroblasts were isolated using collagenase digestion. Some cells were cryopreserved in liquid nitrogen to establish an autologous cell bank. The sample collection process complied with ethical standards.

[0035] Primary fibroblasts that had been frozen were revived and transiently expressed with Yamanaka reprogramming factor to complete partial reprogramming. After reprogramming, karyotype analysis and whole-genome sequencing were performed to confirm the integrity of the cell genome.

[0036] A composite stress was constructed using hydrogen peroxide and tumor necrosis factor-α, and the cells were passaged in 12 rounds across three gradients. In stages one through four, cell viability was the primary screening criterion, eliminating clones with a viability below 40%. In stages five through twelfth, both cell viability and telomere length were used for selection, retaining only clones with a viability of at least 40% and a telomere length of at least 6.5 kb for expansion.

[0037] After domestication, single-cell transcriptome sequencing and whole-genome methylation sequencing were performed on the cells. No tumor suppressor gene inactivation or proto-oncogene abnormal upregulation was detected, and the teratoma formation experiment was negative. The organ pathological sections of the mice in the long-term toxicity test at 6 months showed no abnormalities and no obvious immune rejection reaction.

[0038] Under the same combined stress conditions, the 24-hour in vitro survival rate of the cells of this invention was 82.5% ± 3.1%, while that of the untreated homologous parent cells was only 31.2% ± 2.8%, showing a highly significant difference (P < 0.001). When the cells were injected into aging model mice, and skin collagen was reinfused for 6 months, the youthful group maintained more than 85% of its original level, demonstrating superior results compared to the three control groups.

[0039] Example 1: Transformation after intermediate cryopreservation buffering and acclimatization: Human dermal fibroblasts were cryopreserved in liquid nitrogen for 3 months after lineage resetting treatment, and then acclimatized. Under the same stress, the cell survival rate was 81.9%±2.9%, which was not statistically different from the non-cryopreservation group, and thus fell within the scope of protection of this invention.

[0040] Example 2: Simplified Validation Protocol for Neural Progenitor Cells

[0041] All embryonic neural progenitor cells used complied with relevant ethical guidelines for cell experiments in China. Embryonic neural progenitor cells were harvested, transiently reprogrammed, and subjected to 10 rounds of acclimatization using 1% hypoxia + 5 ng / mL IL-6 + 2 ng / mL TNF-α. Screening thresholds were set at a survival rate ≥50% and telomeres ≥6.5 kb. In a neuroinflammation model under stress for 24 hours, the cell survival rate using this process was 76.3% ± 2.6%, while the survival rate of cells treated with simple reprogramming was only 38.7% ± 3.4%, demonstrating a significant improvement in stress resistance.

[0042] Example 3: Expected Example of Adding Single-Target Preprocessing

[0043] According to the overall process flow of the present invention, CRISPR gene editing is added before the step in Example 1, followed by lineage reset and directed evolution. Based on the general process logic of the present invention, improved cells can be reasonably obtained. This process scheme falls within the protection scope of claims 1 and 11.

[0044] Example 4: Expected Example of Changing Screening Criteria

[0045] By eliminating the dual screening criteria of telomere length and cells, and using only cell proliferation rate as the single screening indicator, while keeping the other processes unchanged, qualified cells can be reasonably obtained based on the general process logic of this invention, falling within the protection scope of claims 1 and 5.

[0046] Example 5: Expected Example of Mammalian Allogeneic Mesenchymal Cells

[0047] After matching and lineage resetting, mouse allogeneic bone marrow mesenchymal stem cells are screened multiple times under metabolic stress, with cell secretory activity as the sole criterion. Under in vitro organ injury simulation stress, based on the general process logic of this invention, a formulation with superior performance to ordinary reset cells can be reasonably prepared, which is suitable for animal organ repair scenarios.

[0048] Comparative example A: Directed evolution only, no lineage reset.

[0049] Homologous fibroblasts were directly screened under stress for 12 rounds, and after 30 rounds of continuous transmission, the posterior granulocyte length was shortened to 4.2kb. Multiple aging-related driver mutations were detected, and the genomic safety was poor.

[0050] Comparative Example B: Lineage reset only, no directed evolution.

[0051] Human dermal fibroblasts underwent lineage resetting without targeted evolutionary screening, and the reset cells were directly reinfused into aging model mice. Within three months of cell reinfusion, skin collagen levels rapidly declined to the baseline levels of the aging model, indicating insufficient long-term improvement.

[0052] Comparative example C: Directed evolution followed by lineage resetting

[0053] First, homologous human dermal fibroblasts underwent 12 rounds of gradient stress-directed evolution screening, followed by lineage reprogramming. The high stress resistance characteristics obtained during screening were completely lost during the reprogramming process. Under the same stress, the cell survival rate was 45.3%±4.2%, which was not significantly different from the cells obtained by simple reprogramming, and the synergistic effect of the technology could not be achieved.

[0054] Industrial applicability

[0055] This invention utilizes conventional cell culture, passage, cryopreservation, and molecular detection equipment, eliminating the need for specialized instruments and raw materials. The finished cell products can be formulated with cryopreservation preparations containing sodium hyaluronate and buffer salts, enabling the creation of injectable cell formulations for in vivo drug delivery and cell repair essences for topical skin application, suitable for both pharmaceutical and cosmetic industrialization. Autologous, allogeneic, human, and mammalian somatic cells can all be mass-produced using this standardized process, resulting in low barriers to industrial adoption.

Claims

1. A method for preparing a cell preparation, characterized in that, Includes the following steps: (a) Somatic cells were subjected to lineage reset treatment to obtain reset cells; (b) Using the reset cells to apply selection pressure for directed evolutionary screening; Step (a) is performed before step (b), or one or more intermediate operations such as cell cryopreservation, thawing, and testing can be set between step (a) and step (b). After the intermediate operation is reset, the cells are still used as raw materials for step (b).

2. The preparation method according to claim 1, characterized in that, The lineage reset process is selected from at least one of induced pluripotent stem cell reprogramming, somatic cell nuclear transfer, partial reprogramming with transient expression of reprogramming factors, and obtaining cells from a pre-established cell bank.

3. The preparation method according to claim 1, characterized in that, The selected pressure includes at least one stress factor.

4. The preparation method according to claim 3, characterized in that, The stress factor is selected from at least one of oxidative stress factors, inflammatory stress factors, metabolic stress factors, hypothermia stress factors, and drug toxicity stress factors.

5. The preparation method according to claim 1, characterized in that, The screening index used in the directed evolution screening is selected from at least one of cell viability, genome stability index, cell proliferation rate, and cell secretory activity.

6. The preparation method according to claim 5, characterized in that, The genome stability index is selected from at least one of telomere length and whole genome methylation profile.

7. The preparation method according to claim 1, characterized in that, The somatic cells are selected from at least one of fibroblasts, neural progenitor cells, mesenchymal stem cells, and immune cells.

8. The preparation method according to claim 1, characterized in that, The directed evolutionary screening employs a gradient-increasing stress level and multiple rounds of passaging and domestication. During screening, a threshold for the screening index is set, and cell clones that reach the threshold are retained.

9. The preparation method according to claim 8, characterized in that, The survival rate threshold is not less than 40%, and the telomere length threshold is not less than 6.5kb.

10. The preparation method according to claim 1 further includes a feedback adjustment step: adjusting the selection pressure intensity of the next batch step (b) based on the batch cell performance test results.

11. The preparation method according to claim 1, characterized in that, Additional steps such as single-target gene editing and small molecule compound treatment can be added.

12. A cell preparation, characterized in that, It is prepared by the preparation method described in claim 1.

13. The cell preparation according to claim 12, characterized in that, The survival rate of the cells under the selected pressure was higher than that of the parental cells that were not treated by the method of claim 1.

14. The application of the cell preparation according to claim 12, characterized in that, The cell preparations are used to prepare drugs that delay or improve age-related degenerative changes.

15. The application of the cell preparation according to claim 12, characterized in that, The cell preparation is used to prepare cosmetics that improve the appearance of aging skin.