CN116847857A

CN116847857A - Methods and systems for increasing stem cell yield

Info

Publication number: CN116847857A
Application number: CN202180093199.5A
Authority: CN
Inventors: 托德·F·奥沃凯蒂斯
Original assignee: Tuo DeFAowokaidisi
Current assignee: Tuo DeFAowokaidisi
Priority date: 2020-12-08
Filing date: 2021-12-08
Publication date: 2023-10-03

Abstract

Platelet rich plasma containing human very small embryonic-like stem cells (hVSEL) is treated with an amplitude modulated pulse of a laser having a predetermined wavelength, wherein the predetermined wavelength ranges from 300nm to 1000nm, for a predetermined period of time. Treatment of platelet rich plasma using this method results in unexpectedly high proliferation of hvsels in the platelet rich plasma when administered to a patient, resulting in a reduction in physiological age.

Description

Methods and systems for increasing stem cell yield

Cross reference

The present application is based on the priority of the following U.S. patent provisional applications, which are incorporated herein by reference in their entirety:

U.S. patent provisional application No. 63/122,831, entitled "Methods and Systems for Increased Production of Stem Cells", filed on 8 th month 12 of 2020;

U.S. patent provisional application No. 63/122,836, entitled "Methods and Systems for Increased Production of Stem Cells", filed on 8 th month 12 of 2020; and

U.S. patent provisional application No. 63/180,742, entitled "Methods and Systems for Increased Production of Stem Cells", filed on 28 days 4 and 4 of 2021;

furthermore, the present application relates to U.S. patent publication No. 20210207121 (U.S. patent application No. 17/146,849) entitled "Methods and Systems for Generation, use, and Delivery of Activated Stem Cells" filed on 1, 12, 2021, which is a continuation of U.S. patent No. 10,907,144 entitled "Methods and Systems for Generation, use, and Delivery of Activated Stem Cells" filed on 2, 2021, and U.S. patent No. 10,907,144 which is a continuation of commonly-entitled issued U.S. patent No. 10,202,598 filed on 12, 2019, U.S. patent No. 10,202,598 which is a continuation of a portion of U.S. patent No. 9,999,785 entitled "Method and System for Generation and Use of Activated Stem Cells" filed on 19, 2018, 6, and U.S. patent No. 9,999,785 which is dependent on priority of U.S. patent provisional application No. 62/006,034 filed on 30, 2014, 5. The' 598 patent also relates to the following U.S. provisional patent applications, which are also incorporated herein by reference in their entirety: U.S. provisional patent application No. 62/321,781, entitled "Method and System for Generation and Use of Activated Stem Cells", filed on day 13, 4 of 2016; and U.S. provisional patent application No. 62/254,220, titled "Method and System for Generation and Use of Activated Stem Cells," filed 11 months 12 days 2015.

The entire contents of the above application are incorporated herein by reference.

Technical Field

Methods and systems for improving stem cell production, particularly using modulated pulses of laser light to increase stem cell proliferation, thereby reversing the biological aging process and/or reducing the physiological age are disclosed.

Background

VSEL (very small embryonic-like) stem cells were first found in mouse bone marrow and were described as small (1-4 μm) non-hematopoietic cells with high nuclear to cytoplasmic ratios. They express surface antigens similar to pluripotent embryonic stem cells. Human VSEL (hVSEL) stem cells were first identified in cord blood and have been shown to be cxcr4+, cd34+, cd133+, oct4+, ssea4+ and lin-, CD45-. The hVSEL stem cells were subsequently demonstrated to be present in peripheral blood and bone marrow, as well as in leukopenia samples collected after granulocyte-colony stimulating factor (G-CSF) administration. The concentration of hVSEL stem cells in peripheral blood is described as 800-1300 cells/mL.

hVSEL stem cells are a population of ectoderm-derived cells produced during embryogenesis of embryonic gastrulations. hVSEL stem cells may be important for long-term production of cd34+ hematopoietic stem cells in bone marrow and may contribute to the repair of experimental Myocardial Infarction (MI). hVSEL stem cells also persist throughout the lifetime in peripheral blood. Thus, it is possible to obtain autologous hVSEL stem cells from any patient of any age, thereby enabling their use in regenerative medicine, simplifying procedures, saving money and reducing adverse reactions associated with allogeneic cells. hVSEL stem cells may also be a viable option for potential pancreatic tissue and human gametes. By proper treatment and administration, the hVSEL stem cells may play a key role in future transformation and regeneration medicine.

Lasers and more generally optical techniques have been used in the stem cell field. For example, 420nm and 540nm laser wavelengths have been shown to stimulate osteogenic differentiation, while other wavelengths do not. Broadband visible light (low level visible light) has been demonstrated to increase proliferation of bone marrow Mesenchymal (MSC) in vitro. The photo-bioremediation of dental pulp MSCs, human fat MSCs and epithelial colony forming units by laser light has also been described.

Flow cytometry is commonly used to evaluate cell proliferation of laser-treated biological samples. Surface antigens Oct 3/4, SSEA4 and CXCR4 in lineage negative (Lin-) compartments were evaluated using flow cytometry. Among these three markers, the endogenous peptide inhibitor EPI-X4, which is known to block binding of flow cytometry antibodies to CXCR4, possibly via its antagonistic ligand. Such blockade of CXCR4 can disrupt or prevent accurate assessment using flow cytometry.

In addition, the organism has a physiological age which is different and independent from the actual age of the organism. The physiological age is measured at the cellular level and may depend on a variety of factors, such as lifestyle, environment, genetics, and the like. The risk of persons of a physiological age to suffer from age-related diseases is lower than the actual age. Techniques for measuring physiological age are well known. In one example, telomere length is used as an indicator of physiological age. In another example, methylation of DNA is assessed, involving a test that determines physiological age by measuring intrinsic epigenetic age; thereby correlating methylation status with physiological age. The age of DNA methylation of embryonic stem cells and induced pluripotent stem cells has been determined to be near zero.

Thus, there is a need for methods of increasing the number of stem cells per volume of Platelet Rich Plasma (PRP) fluid. In particular, methods for increasing proliferation of peripheral blood hVSEL stem cells using modulated laser light bio-modulation are needed. There is also a need for methods of unlocking CXCR4 so that it can readily bind to flow cytometry antibodies. In addition, methods of slowing or reversing the biological clock such that the physiological age is less than the actual age of the organism are desired.

Summary of The Invention

The following embodiments and aspects thereof are described and illustrated in conjunction with systems, tools, and methods, which are meant to be exemplary and illustrative, not limiting in scope. The present specification discloses a number of embodiments.

In some embodiments, the present description relates to a method of reducing the physiological age of a patient comprising: proliferating stem cells of the patient, wherein proliferating includes preparing platelet rich plasma containing the stem cells and treating the platelet rich plasma containing the stem cells with a modulated pulse of laser light having a predetermined wavelength for a predetermined period of time; and administering the treated platelet rich plasma to the patient.

Optionally, the platelet rich plasma is prepared by: adding patient blood to a plurality of tubes; centrifuging the plurality of tubes at a predetermined g-force for a predetermined period of time to produce platelet rich plasma; and aliquoting the resulting platelet rich plasma into sterile tubes.

Optionally, centrifuging the plurality of tubes further comprises shaking the plurality of tubes after centrifuging.

Optionally, the method further comprises shaking the sterile tube after the aliquoting.

Optionally, the plurality of tubes ranges from 3 tubes to 12 tubes, and any increment therein.

Optionally, the method further comprises shaking the platelet rich plasma after treatment with the modulated pulses of laser light.

Optionally, the treatment of the platelet rich plasma is performed under minimal background white light conditions.

Optionally, the predetermined wavelength range is 300nm to 1000nm. Still optionally, the predetermined wavelength is 670nm.

Optionally, normal human blood is used to prepare platelet rich plasma.

Optionally, the predetermined period of time ranges from 1 minute to 5 minutes.

Optionally, the treated platelet rich plasma has a range of 0.5X10 when analyzed immediately after a predetermined period of time ⁶ /mL to 2.0X10 ⁶ Amount of stem cells per mL. Optionally, the treated platelet rich plasma has a range of 0.5X10 when analyzed immediately after a predetermined period of time ⁶ Per mL to 2.0X10 ⁶ Amount of stem cells per mL.

Optionally, the patient experiences a physiological age reduction in the range of 1 year to 4 years of age based on the first administration of the treated platelet rich plasma. Still optionally, the patient experiences a physiological age reduction in the range of 4 years to 9 years based on a second administration of the treated platelet rich plasma. Optionally, the second administration of the treated platelet rich plasma occurs 1 week to 6 months after the first administration.

In some embodiments, the present specification discloses a method of reducing the physiological age of a patient comprising: proliferation of stem cells of a patient, the proliferation of stem cells of a patient comprising: adding normal human blood to the plurality of tubes; centrifuging the plurality of tubes at a predetermined g-force for 10 minutes to produce platelet rich plasma; shaking the plurality of tubes; aliquoting the resulting platelet rich plasma into sterile tubes; shaking the platelet rich plasma in the sterile tube; treating the platelet rich plasma with a modulated pulse of laser light having a predetermined wavelength for a predetermined period of time; shaking the treated platelet rich plasma; and administering the treated platelet rich plasma to the patient.

Optionally, the predetermined period of time ranges from 1 minute to 5 minutes.

Optionally, the treated platelet rich plasma has a range of 0.5X10 when analyzed immediately after a predetermined period of time ⁶ /mL to 2.0X10 ⁶ Amount of stem cells per mL. Optionally, the treated platelet rich plasma exhibits a 2.5-fold increase in stem cells compared to the average of a first control sample and a second control sample, wherein the first control sample comprises platelet rich plasma that has been light treated with a white flashlight light for a predetermined period of time, and wherein the second control sample comprises platelet rich plasma that has not been light treated.

Optionally, modulating eliminates the center wavelength band of the laser so that the remaining upper and lower wavelength bands create a beat pattern of sparse nodes.

In some embodiments, the present specification discloses methods of producing a composition that reduces the physiological age of a patient when administered to the patient, the method comprising: proliferation of stem cells of a patient, the proliferation of stem cells of a patient comprising: preparing platelet rich plasma containing stem cells; and treating the platelet rich plasma with a modulated pulse of laser light having a predetermined wavelength for a predetermined period of time.

Optionally, the platelet rich plasma is prepared using normal human blood.

Optionally, the predetermined period of time ranges from 1 minute to 5 minutes.

Optionally, the treated platelet rich plasma has a range of 0.5X10 when analyzed immediately after a predetermined period of time ⁶ /mL to 2.0X10 ⁶ Amount of stem cells per mL.

In some embodiments, the present specification discloses methods of producing a composition that reduces the physiological age of a patient when administered to the patient, the method comprising: proliferation of stem cells of a patient, the proliferation of stem cells of a patient comprising: adding normal human blood to the plurality of tubes; centrifuging the plurality of tubes at a predetermined g-force for 10 minutes to produce platelet rich plasma; shaking the plurality of tubes; aliquoting the resulting platelet rich plasma into sterile tubes; shaking the platelet rich plasma in the sterile tube; treating the platelet rich plasma with a modulated pulse of laser light having a predetermined wavelength for a predetermined period of time; and shaking the treated platelet rich plasma.

Optionally, the predetermined wavelength range is 300nm to 1000nm. Optionally, the predetermined wavelength is 670nm.

Optionally, the predetermined period of time ranges from 1 minute to 5 minutes.

Optionally, the treated platelet rich plasma exhibits a 2.5-fold increase in stem cells compared to the average of a first control sample and a second control sample, wherein the first control sample comprises platelet rich plasma that has been light treated with a white flashlight light for a predetermined period of time, and wherein the second control sample comprises platelet rich plasma that has not been light treated.

In some embodiments, the present specification discloses a method of reducing the physiological age of a patient, the method comprising: proliferation of stem cells of a patient, the proliferation of stem cells of a patient comprising: preparing platelet rich plasma containing stem cells; and treating the platelet rich plasma with a modulated pulse of laser light having a predetermined wavelength for a predetermined period of time.

Optionally, the platelet rich plasma is prepared by: adding the patient's blood to six tubes; centrifuging the six tubes at a predetermined g-force for a predetermined period of time to produce platelet rich plasma; and aliquoting the resulting platelet rich plasma into sterile tubes. Optionally, centrifuging the six tubes further comprises shaking the six tubes after centrifuging. Optionally, the method further comprises shaking the sterile tube after the aliquoting.

Optionally, the treatment of platelet rich plasma is performed under minimal background white light conditions.

Optionally, the predetermined wavelength range is 300nm to 1000nm.

Optionally, the predetermined wavelength is 670nm.

Optionally, the platelet rich plasma is prepared using normal human peripheral blood.

Optionally, the predetermined period of time is 3 minutes.

Optionally, the treated platelet rich plasma has a concentration of 1.256x10 when analyzed immediately after a predetermined period of time ⁶ Stem cells per mL.

In some embodiments, the present specification also discloses a method of reducing the physiological age of a patient comprising: proliferation of stem cells of a patient, the proliferation of stem cells of a patient comprising: adding normal human peripheral blood into six tubes; centrifuge the six tubes for 10 minutes at a predetermined g-force to produce platelet rich plasma; shaking the six tubes; aliquoting the resulting platelet rich plasma into sterile tubes; shaking the platelet rich plasma in the sterile tube; treating the platelet rich plasma with a modulated pulse of laser light having a predetermined wavelength for a predetermined period of time; and shaking the treated platelet rich plasma.

Optionally, the predetermined wavelength range is 300nm to 1000nm.

Optionally, the predetermined wavelength is 670nm.

Optionally, the predetermined period of time is 3 minutes.

Optionally, the modulation eliminates the center wavelength band of the laser such that the remaining upper and lower wavelength bands create a beat pattern of sparse nodes.

In some embodiments, the present description relates to a method of proliferating stem cells comprising: preparing platelet rich plasma containing stem cells; and treating the platelet rich plasma with a modulated pulse of laser light having a predetermined wavelength for a predetermined period of time.

Optionally, the platelet rich plasma is prepared by: the donated normal human peripheral blood was added to three tubes; centrifuging the three tubes at a predetermined g-force for a predetermined period of time to produce platelet rich plasma; and aliquoting the resulting platelet rich plasma into individual sterile tubes.

Optionally, the predetermined wavelength range is 300nm to 1000nm.

Optionally, the predetermined wavelength is 670nm.

Optionally, the platelet rich plasma is prepared using donated normal human peripheral blood.

Optionally, the predetermined period of time is 3 minutes.

In some embodiments, the present specification discloses methods of proliferating stem cells comprising: the donated normal human peripheral blood was added to three tubes; the three tubes were centrifuged at a predetermined g-force for 10 minutes to produce platelet rich plasma; aliquoting the resulting platelet rich plasma into individual sterile tubes; and treating the platelet rich plasma with a modulated pulse of laser light having a predetermined wavelength for a predetermined period of time.

Optionally, the predetermined wavelength range is 300nm to 1000nm.

Optionally, the predetermined wavelength is 670nm.

Optionally, the predetermined period of time ranges from 1 to 6 minutes. Optionally, the predetermined period of time ranges from 1 to 3 minutes. Optionally, the predetermined period of time is 3 minutes. Optionally, the predetermined period of time is dependent on the volume of platelet rich plasma.

The foregoing and other embodiments of the invention will be described more fully in the accompanying drawings and detailed description provided below.

Drawings

These and other features and advantages of the present specification will be better understood with reference to the following detailed description when considered in conjunction with the accompanying drawings, wherein:

fig. 1 shows a Strachan-ohokaiiys node generator (sonog) device disclosed in U.S. patent No. 6,811,564, the entire contents of which are incorporated herein by reference;

FIG. 2 shows the sparse constructive interference effect of a 1% bandwidth cancellation plate with a 5mm aperture (sparse constructive interference effect);

FIG. 3 is a flow chart showing the steps of a method of preparing a PRP containing hVSEL stem cells according to some embodiments of the present description;

FIG. 4 is a graph showing data for the number and distribution of hVSEL stem cells in untreated PRP;

FIG. 5 shows the results of a typical flow cytometry of PRP without laser treatment;

FIG. 6 is a graph showing data for PRP, costa laser+SONG modulation of related controls, and one day of in vitro culture;

FIG. 7 is a graph showing data relating to Magna Costa laser exposure time variation and SONG modulation variation on day 0 and day 5;

FIG. 8 is a graph showing data relating to Costa laser treatment of hVSEL stem cells in PRP on days 0, 1 and 7;

FIG. 9 is a graph showing data relating to time titration of SONG modulated Magna Costa and Costa lasers against hVSEL stem cells in PRP;

FIG. 10A shows the Intrinsic Epigenetic Age (IEA) of two patients;

fig. 10B shows IEAs for two other patients;

fig. 10C shows IEAs for two other patients;

fig. 10D shows IEAs for two other patients; and

fig. 11 is a flowchart illustrating an exemplary process for preparing a PRP containing hVSEL stem cells for IEA reduction according to some embodiments of the present disclosure.

Detailed Description

The present description is directed to various embodiments. The following disclosure is provided to enable any person of ordinary skill in the art to practice the invention. No language used in the specification should be construed as indicating any non-claimed embodiment as essential to the general practice of the invention or the meaning of the terms used therein. The general principles defined herein may be applied to other embodiments and applications without departing from the spirit and scope of the invention. Also, the terminology and phraseology used is for the purpose of describing the exemplary embodiments and should not be regarded as limiting. Thus, the invention is to be accorded the widest scope encompassing numerous alternatives, modifications and equivalents consistent with the principles and features disclosed. For the sake of clarity, details relating to technical material that is known in the technical fields related to the invention have not been described in detail so as not to unnecessarily obscure the invention.

In the description and claims of the present application, each of the words "comprising," "including," and "having" and forms thereof are not necessarily limited to members of the list associated with those words. It should be noted herein that any feature or component described in connection with a particular embodiment may be used and implemented with any other embodiment, unless explicitly indicated otherwise.

In embodiments, intrinsic Epigenetic Age (IEA) refers to the true physiological age at the DNA level. In embodiments, extrinsic Epigenetic Age (EEA) refers to the immune function status of an organism in addition to other factors that are more sensitive to external factors such as diet, lifestyle, and supplement use.

In embodiments, "normal human blood" is defined as blood that is in a chemical and physical state when immediately drawn from a human body without any further treatment (whether mechanical and/or chemical), also referred to as untreated human blood. As used in this specification, peripheral blood is fluid flowing through the heart, arteries, capillaries, and veins. Its function is to deliver oxygen and other nutrients to cells and tissues of the body and to scavenge carbon dioxide and other waste products from the body. Peripheral blood also plays an important role in the immune system, hormone delivery and temperature regulation.

As used in this specification, platelet Rich Plasma (PRP) may be defined as plasma that has an increased amount of platelets as a result of some form of mechanical and/or chemical treatment relative to plasma that has not undergone such treatment.

SONG device

In various embodiments, to increase yield or proliferation, stem cells are treated with a laser process that exposes the stem cells to a predetermined laser wavelength, a predetermined amplitude modulation, typically on the order of 5x to 10x (although more or less may be used), by a beam expander, and in combination with a device for optical phase conjugation (such as a Strachan-ohokaiiys node generator or a sonog device), which is disclosed in U.S. patent No. 6,811,564 and incorporated herein by reference.

Figure 1 shows a sonog device disclosed in us patent No. 6,811,564. Referring to fig. 1, the sonog device comprises a laser diode 2 controlled by an amplitude modulator 1. The laser diode 2 is chosen to have a substantially linear relationship between current and wavelength with minimal mode hops. The amplitude modulator 1 modulates the current to the laser diode 2, which in turn results in a very small wavelength modulation of the laser for the purposes discussed below.

The output of the laser diode 2 is collimated by a lens 3 and passed to an optical element 4. The optical element 4 consists of a first diffraction grating, a refractive element and a second diffraction grating such that the light beam is substantially cancelled. This allows cancellation to occur over a small portion of the laser source wavelength variation, rather than over a single critical wavelength. Wavelengths above and below the center frequency that exceed the acceptance bandwidth of the cancellation optics 4 pass without being cancelled. This means that a complex Fresnel/fraunhofer region is created, defined by the beat frequencies of the high and low frequencies as a function of the aperture. Thus, in a selected direction from the aperture, a relatively sparse constructive interference zone occurs between the high frequency channel and the low frequency channel of the cancellation element, as shown in fig. 2. Fig. 2 shows the sparse constructive interference effect of a 1% bandwidth cancellation plate of a 5mm aperture. Black represents a constructive node.

As shown in fig. 1, the optical element 4 can be angularly adjusted between positions 4A and 4B. This changes the ratio of constructive to destructive interference. In addition, in embodiments, the system of fig. 1 may include a mechanism for aligning the composite beam exiting from the optical element 4 with the collimated beam exiting from the collimator 3.

In practice, a continuous beam is converted into a train of very short duration pulses, typically of about a sub-femtosecond duration. The small wavelength modulation of the laser diode 2 results in the constructive and destructive nodes moving rapidly through the volume of the fresnel zone of the collimator lens aperture. This has the effect of stimulating very short (sub-picosecond) pulse behavior at any point in the fresnel zone where the node passes through the fresnel zone at a pulse repetition frequency defined by the amplitude modulator frequency.

The wavelength of the cancellation and constructive interference regions of a single path will theoretically be the difference between the two frequencies. If the bandwidth of the cancellation element is very narrow, this difference is very small and the effective wavelength of the cancellation/non-cancellation period will be very long, on the order of picoseconds. Thus, the system behaves substantially like a system without cancellation because it requires a much larger aperture than the primary wavelength to create a useful fresnel/fraunhofer region. Such apertures will greatly increase the available fischer-tropsch path, eliminating any useful impact, even though it is possible to generate a source of sufficient coherence for such apertures.

If the beat frequency can be high enough, the wavelength of the cancellation to non-cancellation period can be a fraction of the actual aperture. This will be small enough to limit the ferman path to one or two periods in free space, thereby making the fresnel/fraunhofer effect apparent. Since the center frequency and spectral spread of the laser diode are modulated by adjusting the junction current and/or temperature, the fresnel/fraunhofer pattern varies significantly with very small variations in the wavelength of one or both of the pass frequencies. Such modulation is produced by the amplitude modulator 2 in the apparatus of fig. 1.

Conventional coherent or incoherent light beams have high probability paths in the fischer-tropsch plot. These paths overlap at very low frequencies (kHz) and have little practical use in stimulating molecular resonance. It should be noted, however, that the above phenomenon is used as a means of multiplying the modulation frequency up to the point where the light beam effectively becomes continuous. Thus, by properly selecting the aperture, the area of the beam selected for transmission through the medium, and the modulation frequency, the constructive node can be made to pass through any given point in the beam at a frequency many times higher than the modulation frequency. Under ideal conditions, the duration of the constructive node exposed to any point will correspond to a quarter of the wavelength duration of the molecular frequency repeated once per cycle.

If the wavelength of the laser is selected to be a wavelength that is easily absorbed by the atomic structure that is desired to induce resonance, the beam will effectively deliver the desired modulation frequency to the desired molecule. Cell adhesion molecules and human integrins (such as α4 and β1) are well suited for excitation to chemical activity by this method.

The cell source used in the procedures described herein may be autologous or exogenous. Autologous stem cells refer to cells derived from the same person to be treated with such cells. These cells will be genetically matched, thereby eliminating the risk of cell rejection. In the current methods, autologous stem cells are derived or concentrated from peripheral blood, bone marrow or fat, but other tissues may also be sources of autologous stem cells, as virtually every tissue of the body has its own unique stem cell bank.

A preferred exogenous source of stem cells is cord blood. Stem cells from cord blood are very powerful, with long telomeres (the level of the genetic senescence clock at the neonatal level) and powerful tissue repair capacity. Functionally, in the case of the complete immune system, the cell rejection syndrome and Graft Versus Host Disease (GVHD) are not problems with the use of such cell sources. Matched bone marrow may also be the source of cells, but a high degree of matching is required to avoid rejection and GVHD. In practice, umbilical cord blood stem cells have been safely used for regeneration, as opposed to anti-leukemia medical regimens.

Preparation of Platelet Rich Plasma (PRP) containing hVSEL stem cells

Fig. 3 is a flow chart illustrating a method of preparing PRP containing hVSEL stem cells according to some embodiments of the present disclosure. Anticoagulated (sodium citrate) donated normal human peripheral blood (450 mL) was obtained and stored at 4 ℃ prior to use. The blood was warmed to room temperature prior to treatment for PRP.

In step 302, 11mL of whole peripheral blood (normal human blood) was added/aliquoted into three PRP tubes by obtaining each PRP sample using three PRP tubes. At step 304, the tube containing whole blood PRP is centrifuged at a preset g-force for 10 minutes. As a result, each of the three PRP tubes containing 11mL of whole blood produced about 6mL (about 18mL total) of PRP.

At step 306, the PRP produced at step 304 is aliquoted into individual sterile tubes for further manipulation and analysis using sterile techniques in a class II flow hood. Each 18mL PRP formulation was generated in triplicate for each procedure and evaluation.

Processing, modulation, manipulation and assessment of human PRP containing hVSEL stem cells

According to aspects of the present description, a laser pulse having a wavelength in the range of 300nm to 1000nm, and in one embodiment about 670nm, is used to manipulate or modify PRP containing hVSEL stem cells. In some embodiments, PRP containing hVSEL stem cells is manipulated or modified using two lasers:

Costa laser: in one embodiment, the Costa laser employed is a 670nm, 5mW SONG modulated laser. In embodiments, the level of Optical Phase Conjugation (OPC) varies for experimental purposes. In embodiments, the level of optical phase conjugation is in the range of 1% to 99%. In one embodiment, the sonog modulated laser is set to 60% Optical Phase Conjugation (OPC) to obtain a resulting beam power of 1 mW.

Magna Costa laser: in one embodiment, the Magna Costa laser used is a 670nm, 5.7mW SONG modulated laser. In embodiments, the level of Optical Phase Conjugation (OPC) varies for experimental purposes. In embodiments, the level of optical phase conjugation is in the range of 1% to 99%. In one embodiment, the sonog modulated laser is set to 60% opc to obtain a resulting beam power of 1.3 mW. The Magna Costa laser has an adjustable waveform, and alternative waveforms can be introduced as controls.

In an embodiment, the sonog modulation of the laser eliminates the center wavelength band of the laser output due to non-fringe destructive interference. The remaining upper and lower wavelength bands create beat patterns of the constructive interference sparse nodes, which represent the remaining physical visible light. This modulation of the complex waveform pattern results in rapid shifting of the nodes, which can achieve pulse repetition frequencies of every femtosecond or less. Destructive interference and sparsity of the nodes reduces flare (flare) of the tissue interface surface. This reduces the reflectivity of photons entering the region that has just undergone photon absorption as well as the scattering effect. The penetration depth of the sparse node on the surface of the interface of human skin and the like can be 10-20 times of that of common photons.

Incubation and harvesting of laser-treated and control (no laser or white light) hvsels in PRP

In some embodiments, to assess the biostability of the effects of laser exposure or manipulation, PRP is cultured in an equal volume of RPMI 1640 medium supplemented with 200mM L-glutamine, penicillin and streptomycin, and 10% heat inactivated fetal bovine serum.

All PRP cultures were performed in a humidified incubator set at 37℃with 5% CO in air using a T25 vented flask ₂ . If necessary, adherent cells were harvested by initial washing with Dulbecco PBS without Ca2+/Mg2+ and treatment with trypsin EDTA at 37℃for 5 min.

Number and distribution of hVSEL stem cells in untreated PRP

In one embodiment, to assess the distribution and number of hVSEL stem cells in untreated PRP tubes, PRPs were separated into discrete sample tubes after centrifugation by the following steps (see flow chart of fig. 3): taking 2mL of the "top" portion of PRP; 2mL of the "middle" portion of PRP; the "bottom" portion of 2ml rp-as close as possible to the red blood cell interface; 2mL red blood cell fraction top; 2mL red blood cell fraction bottom, total 5 tubes per PRP sample. The number of hVSEL stem cells per sample was assessed using the flow cytometry protocol previously mentioned in this specification. Evaluation of each sample indicated that cell viability remained at >90%.

Fig. 4 is a graph 400 showing data on the number and distribution of hVSEL stem cells in untreated PRP in each fraction obtained in the method described with respect to fig. 3. The average hVSEL stem cell count of 2mL at the top of PRP was found to be 3.1X10 ⁵ Per mL, average Lin-cell count of 20.0x10 ⁵ /mL. The average hVSEL stem cell count of 2mL in the middle of PRP was found to be 4.27x10 ⁵ Per mL, average Lin-cell count was 18.5X10 ⁵ /mL. The average hVSEL stem cell count at the bottom 2mL of PRP was found to be 9.29x10 ⁵ Per mL, average Lin-cell count of52.2x10 ⁵ /mL. The total average number of hVSEL stem cells found in PRP was 1.66x10 ⁶ /mL. The total average number of Lin-cells found in PRP was 9.01x10 ⁶ /mL. The total number of hVSEL stem cells in the top fraction of erythrocytes was 4.0x10 ² Per mL, average Lin-cell count of 1.65x10 ⁴ /mL. The total number of hVSEL stem cells in the bottom fraction of erythrocytes was 6.0x10 ² Per mL, average Lin-cell count of 5.1X10 ⁴ /mL。

In this embodiment, it is shown that there is an average of 1.6X10 in PRP obtained from donated human blood ⁶ hVSEL stem cells/mL. By taking an average of the untreated values of PRP for all the different extracts, it is also possible to provide another average estimate of total hVSEL stem cells/mL in PRP. The resulting average value was 3.92x10 ⁶ /mL. In this gradient study, hVSEL stem cells were observed in the PRP normal peripheral blood (normal human blood) in the range of 0.746-16x10 ⁵ /mL。

PRP was found to have a gradient of hVSEL stem cells from the top meniscus of PRP down to the PRP/red blood cell interface where the highest number of hVSEL stem cells was found, indicating that the full volume of PRP should be used in some embodiments to obtain the best results. Thus, multiple volumes of PRP were pooled for clinical use. In embodiments, some applications may require higher hVSEL concentrations per volume PRP. For example, in some topical applications, such as hair and cosmetic applications, it is desirable to have a higher concentration of hVSEL. In these cases, the presence of up to the bottom third of the hVSEL cells can be used to obtain a more concentrated effect. In other applications, such as systemic treatment, which may be administered intravenously, it may be desirable to combine full volumes.

Few hVSEL stem cells in the red blood cell fraction of PRP tubes (about 1x10 ³ /mL). These data demonstrate that PRP-based isolation of hVSEL stem cells works very effectively when using the systems and methods of the present description. Fig. 5 shows the results 500 of a typical flow cytometry for PRP without laser treatment.

Laser treatment of PRP (using Costa laser) and proliferation of resulting hVSEL stem cells on day 0 and day 1 of culture

In one embodiment, to assess the effect of using a SONG modulated Costa laser on the number of hVSEL stem cells in a PRP, the PRP was prepared in triplicate as previously described with reference to FIG. 3. The first batch was exposed to Costa laser + SONG (set to 60% OPC) light for 3 minutes, the second batch was exposed to white flashlight light for 3 minutes, and the third batch received no treatment (control). After flow cytometry analysis, three PRP samples were cultured and then collected on day 1 for flow cytometry analysis. The purpose of this embodiment was to evaluate the initial effect of the laser on the proliferation of hVSEL stem cells and to see if these changes were stable after 24 hours of in vitro culture. Others have described up-regulation of genes in human dermal cells following laser exposure, resulting in increased paracrine secretion.

Figure 6 is a graph 600 showing data on Costa laser + SONG modulation of PRP, related controls, and in vitro culture for one day. As shown, when PRP was treated with laser +SONGs for 3 minutes and then immediately analyzed by flow cytometry, the number of hVSEL stem cells was 1.256×10 ⁶ /mL. The same batch of PRPs treated with white flashlight light for 3 minutes (as a first control) and analyzed immediately contained 4.15X10 ⁵ /mL hVSEL stem cells. The same batch of untreated PRP (as a second control) contained 5.77x10 ⁵ /mL hVSEL stem cells. The average of these two control samples was 4.96x10 ⁵ /mL. Thus, laser-exposed PRP showed an increase in hVSEL stem cell number by 2.5X (2.5 fold) compared to the average of the two control groups. This is a rapid effect because cells are removed for analysis on a flow cytometer immediately after modulated laser exposure. Thus, in any study, the time from modulated laser exposure to flow cytometry analysis did not exceed 30 minutes. This observation is compared to clinical use and clinical trials of modulated laser exposed hVSEL in PRP, which generally shows rapid clinical improvement after intravenous infusion of autologous laser exposed hVSEL stem cells in PRP. This is the first time these laboratory observations are correlated with clinical data.

In embodiments, it should be noted that PRP may be treated with laser + sonog for 1 to 5 minutesA predetermined period of time within the range, and preferably 3 minutes. In an embodiment, the treated platelet rich plasma has a range of 0.5X10 when analyzed immediately after a predetermined period of time ⁶ /mL to 2.0X10 ⁶ Amount of stem cells per mL. In embodiments, the number of treated hVSEL stem cells is 1.256×10 ⁶ /mL。

On day 1 of in vitro culture (i.e., after 24 hours of in vitro culture), the Costa laser + SONG treated PRP contained 1.086x10 ⁵ /mL hVSEL stem cells. On day 1 of in vitro culture, white torch PRP (first control) contained 0.447X 10 ⁵ /mL hVSEL stem cells. On day 1 of in vitro culture, control PRP (untreated and second control) contained 0.376x10 ⁵ /mL hVSEL stem cells. The average value of the two control groups was 0.432×10 ⁵ /mL. After 24 hours in vitro, laser-exposed PRP showed an increase in hVSEL stem cells by 2.5X (2.5 fold) over control cells, indicating that the ratio of laser-modulated hVSEL stem cells to control hVSEL stem cells remained unchanged for 24 hours even though the actual cell count was reduced (as expected after in vitro culture). In embodiments, stem cell administration occurs within a time range of 1 minute to 24 hours of preparation/laser modulation. In embodiments, stem cell administration occurs within a time range of 1 minute to 2 hours of preparation/laser modulation. In embodiments, stem cell administration preferably occurs within 30 minutes of preparation/laser modulation.

These data demonstrate that laser light has a proliferative effect on hVSEL stem cells in PRP. This effect remained relatively horizontal for at least 24 hours in vitro after laser exposure. In embodiments, stem cell administration can occur within a time frame that the measurable effect on the hVSEL remains unchanged after laser exposure, wherein the time can vary according to a variety of conditions.

Laser treatment of hVSEL stem cells in PRP (using Magna Costa laser), titration of laser exposure time and ± SONG modulation on day 0 and day 5

In one embodiment, hVSEL stem cells in PRP were treated with Magna Costa laser to assess the amount of hVSEL present in PRP with and without sonog modulation 1-3 minutes after laser exposure to confirm optimal settings for clinical use. As previously described in this specification, the Magna Costa laser is identical to Costa, except that the waveform is tunable. This enables the use of potentially improved "flat" wave control in these experiments.

In this embodiment, the sonog modulation was set to 60% OPC, and all cells were analyzed on day 0, then cultured in vitro for 5 days to assess the persistence of any proliferation changes in hVSEL.

The purpose of this embodiment is to assess the effect of laser exposure time and the application of sonog modulation or no sonog modulation on hVSEL stem cell proliferation in PRP on the day of laser exposure (D0) and five days after in vitro (D5). Laser exposure time and sonog modulation are critical to successful proliferation of hVSEL stem cells.

Fig. 7 is a graph 700 showing data relating to Magna Costa laser exposure time variation and sonog modulation variation on days 0 and 5. In embodiments, the laser exposure time ranges from 1 minute to 6 minutes. In embodiments, the laser exposure time ranges from 1 minute to 3 minutes. In embodiments, the laser exposure time is 3 minutes for volumes ranging from 20 milliliters to 30 milliliters. In other embodiments, the laser exposure time depends on the volume of the PRP. In other embodiments, the laser exposure time depends on the quality of the PRP harvested.

As shown, on day 0 (the day when PRP was prepared and laser irradiated), the total number of hVSEL stem cells in PRP increased with increasing laser irradiation time (from 1 minute to 3 minutes), and sonog modulation was present throughout. The 2 and 3 minute laser exposure times produced very similar numbers of hVSEL stem cells. There is a similar but less pronounced increase in the number of hVSEL stem cells when the laser is applied without sonog modulation. The flat wave and untreated controls remained similar, and it should be noted that the flat wave laser exposure time was 3 minutes.

Thus, in PRP exposed to sonog modulated Magna Costa laser for 1, 2 and 3 minutes, the number of hVSEL stem cells was highest in 2 and 3 minute treatments. There were fewer hVSEL stem cells in the Magna Costa lasers without sonog modulation than the laser sonog modulation group in 1, 2 and 3 minutes, but the detected hVSEL stem cells steadily increased during the laser exposure time. Sonog modulated Magna Costa flat wave and no treatment control (hVSEL number) were lower than equivalent sonog modulated laser cell counts at 2 and 3 minutes laser exposure.

On day 5 of in vitro culture, the sonog modulated laser group showed an increase in the number of hVSEL stem cells compared to day 0, with slightly more hVSEL stem cells present during the 2 and 3 minute laser exposure times. The 1 minute and 3 minute laser exposures without sonog modulation contained more hVSEL stem cells than the 2 minute laser exposure, and the flat wave and no treatment controls also contained more hVSEL stem cells overall than day 0.

Thus, after 5 days of in vitro culture, both 5 day hVSEL stem cell counts showed an increase in hVSEL stem cells compared to day 0. The increase in the control group was also greater than the increase in the experimental group. This abnormality requires further investigation, as it may be a true reflection of the proliferation of hVSEL stem cells in vitro, or it may be simply an abnormality in this particular embodiment. In general, when laser treated hVSEL stem cells are cultured in vitro, a decrease in cell number is observed.

Costa laser treatment (±song modulation) of hVSEL stem cells in PRP on day 0, day 1 and day 7

In one embodiment, PRP (prepared according to the method of fig. 3) is exposed to Costa laser for 3 minutes in the case of SONG modulation, and for 3 minutes in the absence of SONG modulation. The resulting PRP was then assessed for hVSEL proliferation and then placed in vitro for 1 and 7 days. Cultures were harvested on day 1 and day 7 and the resulting cell harvest was evaluated for hVSEL proliferation using flow cytometry. This embodiment also includes an assessment of the amount of hVSEL in whole peripheral blood after erythrocyte lysis.

This embodiment involves assessing the number of hVSEL stem cells in PRP on the day of laser treatment and on day 1 and day 7 of cell in vitro culture, and assessing the effect of laser treatment with and without sonog modulation. The number of hVSEL stem cells in peripheral blood was measured without any treatment. This involves erythrocyte lysis followed by flow cytometry.

Fig. 8 shows a graph 800 showing data relating to Costa laser treatment of hVSEL stem cells in PRP on days 0, 1 and 7. The number of hVSEL stem cells in the peripheral blood sample was 8.1X10 ⁵ Per mL, which is 1x10 compared to the number of hVSEL stem cells in the PRP estimated previously ⁶ The number of hVSEL stem cells in the PRP control of this study was 1.072x10 per mL ⁶ the/mL is closely related. It is expected that the hVSEL stem cell count in PRP will be slightly higher than peripheral blood because hVSEL stem cells are concentrated in PRP.

After 3 minutes of SONG modulated laser treatment, the number of hVSEL stem cells in PRP increased to 2.22x10 ⁶ Per mL, day 1 of culture was 7.82x10 ⁵ Per mL, and on day 7 of culture was 2.56x10 ⁵ /mL. After 3 minutes of unmodulated laser treatment, the number of hVSEL stem cells in PRP increased to 1.994x10 ⁶ Per mL, 1.348X10 on day 1 of culture ⁶ Per mL, and on day 7 of culture 1.48x10 ⁵ /mL。

After 3 minutes of white light treatment, the number of hVSEL stem cells in PRP increased to 1.504×10 ⁶ Per mL, culture day 1 was 2.66x10 ⁶ Per mL, and on day 7 of culture was 2.18x10 ⁵ /mL. After no treatment (as control), the number of hVSEL stem cells in PRP was 1.072x10 ⁶ Per mL, 4.7X10 on day 1 of culture ⁵ Per mL, and on day 7 of culture 1.657x10 ⁵ /mL。

This embodiment demonstrates the presence of hVSEL stem cells in whole peripheral blood after erythrocyte lysis. The data show an increase in the number of hVSEL stem cells in PRP, which demonstrates that PRP is an effective way to isolate hVSEL stem cells for experimental and clinical use.

The maximum number of hVSEL stem cells in PRP was found with a Costa laser modulated with SONG with an exposure time of 3 minutes. The same laser exposure without sonog modulation showed fewer hVSEL stem cells, but the level was still increased compared to the control group, indicating that laser exposure may bring some benefit even without sonog modulation. Both the white light and the no-treatment control showed fewer hVSEL stem cells than the sono-modulated and sono-unmodulated treatments.

The reduced number of hVSEL stem cells occurring after 1 day and 7 days of in vitro culture may reflect the cell death associated with in vitro culture.

Time titration of hVSEL stem cells in PRP by SONG modulated Magna Costa and Costa lasers

In one embodiment, PRP (prepared according to the method of fig. 3) was exposed to Magna Costa laser for 3 minutes and Costa laser for 3, 6, and 9 minutes. White light control and no treatment control were used. Thereafter, hVSEL stem cell flow cytometry analysis was performed for all exposure times.

This embodiment aims to identify the optimal laser exposure time for hVSEL stem cell proliferation in PRP. Fig. 9 shows a graph 900 showing data relating to time titration of sonog modulated Magna Costa and Costa lasers to hVSEL stem cells in PRP. In embodiments, the laser exposure time ranges from 1 minute to 6 minutes. In embodiments, the laser exposure time ranges from 1 minute to 3 minutes. In embodiments, the laser exposure time is 3 minutes for volumes ranging from 20 milliliters to 30 milliliters. In other embodiments, the laser exposure time depends on the volume of the PRP. In other embodiments, the laser exposure time depends on the quality of the PRP harvested. As shown, the total number of hVSEL stem cells found in PRP exposed to SONG modulated Costa Magna and Costa laser for three minutes was higher than that of the 6 or 9 minutes of exposure to SONG modulated Costa laser. These data confirm that the optimal laser exposure time to maximize hVSEL stem cell proliferation is 3 minutes. White light (flashlight) control and no-treatment control showed that the hVSEL stem cell number was less than 3 minutes of sonog modulated laser exposure, confirming the optimal exposure time was 3 minutes.

The data from the various embodiments of the present specification demonstrate that laser treatment, exposure, or modulation of hVSEL stem cells in PRP results in proliferation of hVSEL stem cells. This has great potential in the routine treatment of the future and in understanding the true nature of hVSEL stem cells.

In embodiments, optimization of PRP formulations for laser-activated hVSEL stem cells depends on a number of factors including, but not limited to, centrifugation time, cell collection, laser treatment, and time between patient administrations. In addition, in embodiments, a three shake method may be employed that may a) result in increased yields of cells concentrated at the interface between the plasma and gel where separation is achieved, less loss of cells due to adhesion to the interface, and b) increase in cytokines and growth factors present in the formulation before or after laser treatment.

Unlocking CXCR4 to make it available for binding

Endogenous peptide inhibitor X4 (EPI-X4) is an antagonistic ligand for CXCR 4. Such naturally occurring peptides are derived from fragments of albumin, which bind to CXCR4 antigen, primarily through interactions of their N-terminal residues in the small pocket of CXCR4, thereby inhibiting G protein signaling to the relevant cells. Several derivatives of EPI-X4 have been reported, whose IC50 values indicate that the N-terminal residue of EPI-X4 is critical for binding to CXCR 4.

Subsequent studies have shown that the NTer-IN configuration (N-terminal of EPI-X4 is located IN the small pocket of CXCR 4) plays a crucial role IN CXCR4/EPI-X4 binding. Furthermore, only seven EPI-X4 residues play an important role in this binding, four of which are positively charged, interacting through the small pocket of CXCR 4.

Furthermore, the negatively charged EPI-X4 residue L16 (C-terminal Leu) interacts with CXCR4 residue K271 (Lys) with destabilizing effects. However, chemical elimination of L16 has little effect on EPI-X4 binding to CXCR4, suggesting that the first three salt bridges and hydrogen bonds are the major factors of binding.

The last two of the seven important interactions, V11 and T15 of EPI-X4, interact with E25 and R30 (both constituting the β chain of CXCR 4), also providing some small additional binding stability. Chemical elimination of EPI-X4 residues L1 or K7 almost completely eliminates receptor binding.

Salt bridges are interactions of electrostatic bonding and hydrogen bonding between oppositely charged residues. Although hydrogen bonds can combine to produce a major force as in water, individual bonds are weak and are prone to breakage. The distance between residues involved in the salt bridge is important, typically on the order of <400 picometers (pm). Amino acids above this distance do not meet the conditions for formation of salt bridges and salt bridges may undergo thermal fluctuations, thereby constantly breaking and reforming hydrogen bonds.

EPI-X4 is derived from albumin fragmented under acidic conditions of embryonic gastrulation, binds to and deregulates CXCR4 expressed by hVSEL stem cells, protecting salt bridges and hydrogen bonds in the CXCR4 small pocket from thermal fluctuations, thus maintaining hVSEL stem cells stationary.

CXCR4 is not blocked by the sonog modulated laser, making it easily bound by flow cytometry antibodies. The sonog modulated red laser penetrates the small pocket of CXCR4, disrupting the hydrogen bonds and salt bridges that bind CXCR4 to EPI-X4. It was observed that sonog modulated laser exposure for three minutes was most effective for unlocking CXCR 4. Laser thermal fluctuations in the CXCR4 small pocket can maximize in vitro proliferation of hVSEL stem cells over a given time. Within three minutes, the bond of EPI-X4 to CXCR4 was broken and the laser became ineffective as the thermal energy of the small pocket was comparable to that of the red energy laser.

After three minutes of continuous laser exposure, as the fluctuating conditions tend to calm, new thermal stability builds up and new hydrogen bonds (if not salt bridges) form under hotter but currently stable conditions. When laser irradiated for six and nine minutes, the hVSEL count decreased because the hotter stable conditions in the CXCR4 small pocket allowed some new hydrogen bonds to occur, which clearly showed some recombination effects on the CXCR4/EPI-X4 complex.

The apparent rapid proliferation of hVSEL stem cells in PRP in vitro suggests that three minutes of sonog modulated red laser penetration into the small pocket of CXCR4 and breaking the salt and hydrogen bridges, disrupting CXCR4/EPI-X4 binding and exposing CXCR4 to labeled antibodies in subsequent flow cytometry analysis.

Intrinsic age reduction

Intrinsic Epigenetic Age (IEA) is a true indicator of physiological age at the DNA level. In one embodiment, a single treatment as described herein may reduce the IEA of an individual by 2 to 4 years old, a second treatment may reduce the IEA of an individual by 2 to 4 years old, a third treatment may reduce the IEA of an individual by 2 to 4 years old, and a fourth treatment may reduce the IEA of an individual by 2 to 4 years old, using the treatments and administration procedures described herein. Thus, for each treatment, IEA may be reduced by 2 to 4 years of age, and thus, four treatments over a period of 1 to 24 months may reduce IEA in an individual by 8 to 16 years of age. As another example, an individual's IEA may be reduced in the range of 1 to 4 years of age using the treatment and administration procedures described herein. More specifically, in one embodiment, a single treatment as described herein may reduce the individual's IEA by 1 to 4 years old, a second treatment may reduce the individual's IEA by an additional 1 to 5 years old, a third treatment may reduce the individual's IEA by an additional 1 to 5 years old, and a fourth treatment may reduce the individual's IEA by an additional 1 to 5 years old. Thus, for each treatment, IEA may be reduced by 1 to 5 years of age, and thus, four treatments over a period of 1 to 24 months may reduce IEA in an individual by 4 to 20 years of age. In embodiments, the treatment is administered weekly to annually and in any increment therein. In embodiments, the treatment is administered weekly to every six months and in any increment thereof. Optionally, the treatment may be administered at any frequency, so long as it achieves the objects of the present specification.

In embodiments, the patient experiences a physiological age reduction in the range of 1 year to 4 years based on the first administration of the treated platelet rich plasma. In embodiments, the patient experiences a physiological age reduction in the range of 4 years to 9 years based on a second administration of the treated platelet rich plasma. In embodiments, the second administration of the treated platelet rich plasma occurs 1 week to 6 months after the first administration.

10A-10D, examples of intrinsic epigenetic ages determined for eight different people (referred to as patient A-H) are provided. Fig. 10A shows that IEA of patient a 1010 is at 50.44 years old, with the actual age of patient a being 57 years old. The figure also shows that IEA of patient B1020 is at 50.34 years old, with the actual age of patient B being 62 years old. Fig. 10B shows that IEA of patient C1030 is 51.87 years old, with patient C having an actual age of 50 years. The figure also shows that IEA of patient D1040 is at 42.98 years old, with the actual age of patient D being 42 years old. Fig. 10C shows that IEA of patient E1050 is at 37.10 years old, with patient E having an actual age of 36 years. The figure also shows that the IEA of patient F1060 is at 47.03 years, when the actual age of patient F is 53 years. Fig. 10D shows that IEA of patient G1070 is at 63.41 years old, with the actual age of patient G being 66 years old. The figure also shows that the IEA of patient H1080 is at 50.62 years, at which time the actual age of patient H is 50 years. Thus, the actual age may be very different from the physiological age, which may be further different for IEA and EEA.

In general, it is desirable to reduce the rate of epigenetic aging, particularly IE aging, inhibit or even reverse epigenetic aging. For this, each patient was administered with the treatment as shown in fig. 11. Fig. 11 is a flowchart illustrating an exemplary process for preparing a PRP containing hVSEL stem cells for IEA reduction according to some embodiments of the present disclosure. IEA is reduced by increasing regenerative growth factors produced by proliferation of hVSEL stem cells. In step 1102, the patient's blood is obtained in a plurality of 10cc tubes each. In embodiments, the number of tubes ranges from 3 to 12. In a preferred embodiment, the patient's blood is obtained in six tubes of 10cc each. In step 1104, each tube is rotated with a centrifugal G force of about 270G for about 10 minutes. This spinning process pulls red/white blood cells into the gel at the bottom of each tube. Platelets, hVSEL stem cells, and plasma remain separated above the gel in the form of PRP. According to the basal density profile, the upper third of PRP had the lowest concentration of hVSEL stem cells, while the lower third of PRP near the gel interface had the highest concentration of hVSEL stem cells. At step 1106, the tube is first rocked. Shaking included gently shaking the tube back and forth for about 10 seconds. This action knocks loose hVSEL stem cells in PRP near the gel boundary, increasing hVSEL yield by at least 1% compared to the same procedure without such shaking. At step 1108, about 6 to 7cc of PRP is harvested per tube. The harvested amounts were collected in separate sterile tubes. At step 1110, the tube containing the PRP harvested at step 1108 is shaken (second shake). In some embodiments, vigorous shaking is performed for about 20 seconds to release the rejuvenating factors. At step 1112, laser stimulation is applied. Laser stimulation was applied as described above. In some embodiments, the sono-modulated laser stimulus is applied for three minutes. At step 1114, the tube is shaken a third time to wake up dormant hVSEL stem cells and to obtain cytokines and growth factors from the hVSEL stem cells.

Fig. 11 provides an exemplary process for processing PRP obtained from a blood sample of a patient. Variations in the method may also reduce IEA without departing from the scope of the invention. The decrease in physiological age may increase on a per treatment basis. The treatment depicted in fig. 11 is repeated to achieve a further reduction in physiological age. Thus, in one example, one treatment may reduce the physiological age by one year, while additional treatments may reduce it by an additional year. The IEA reduction achieved by embodiments of the present description is higher than any other known treatment.

Thus, returning to the case examples provided in the illustrations of fig. 10A-10D, if patient a is provided with one treatment, the actual age decreases from 50.44 years old to about 48 to 46 years old. If patient B is provided with two treatments, separated by a period of 1 week to 6 months, the actual age decreases from 50.34 years to about 46 to 42 years. If patient C is provided with three treatments, each separated by a period of 1 week to 6 months, the actual age decreases from 51.87 years to about 46 to 40 years. If patient D is provided with four treatments, each separated by a period of 1 week to 6 months, the actual age decreases from 52.98 years old to about 45 to 37 years old. If patient E is provided with five treatments, each separated by a period of 1 week to 6 months, the actual age decreases from 37.10 years old to about 27 to 17 years old. If patient F is provided with six treatments, each separated by a period of 1 week to 6 months, the actual age decreases from 47.03 years to about 35 to 23 years. Finally, if patient G is provided with seven treatments, each separated by a period of 1 week to 6 months, the actual age decreases from 63.41 years to about 49 to 35 years.

The above examples are merely illustrative for many applications of the system of the present invention. Although only a few embodiments of the present invention have been described herein, it should be understood that the present invention may be embodied in many other specific forms without departing from the spirit or scope of the present invention. The present examples and embodiments, therefore, are to be considered as illustrative and not restrictive, and the invention may be modified within the scope of the appended claims.

Claims

1. A method of producing a composition that reduces the physiological age of a patient when administered to the patient, the method comprising:

proliferating stem cells of the patient, comprising:

preparing platelet rich plasma containing stem cells; and

the platelet rich plasma is treated with a modulated pulse of laser light having a predetermined wavelength for a predetermined period of time.

2. The method of claim 1, wherein preparing the platelet rich plasma comprises:

adding the patient's blood to a plurality of tubes;

centrifuging the plurality of tubes at a predetermined g-force for a predetermined period of time to produce the platelet rich plasma; and

the resulting platelet rich plasma was aliquoted into sterile tubes.

3. The method of claim 2, wherein said centrifuging the plurality of tubes further comprises shaking the plurality of tubes after centrifuging.

4. The method of claim 2, further comprising shaking the sterile tube after aliquoting.

5. The method of claim 2, wherein the plurality of tubes ranges from 3 tubes to 12 tubes, and any increments therein.

6. The method of claim 2, further comprising shaking the platelet rich plasma after treatment with the modulated pulses of laser light.

7. The method of claim 1, wherein said treatment of said platelet rich plasma is performed under minimal background white light conditions.

8. The method of claim 1, wherein the predetermined wavelength range is 300nm to 1000nm.

9. The method of claim 1, wherein the predetermined wavelength is 670nm.

10. The method of claim 1, wherein the platelet rich plasma is prepared using normal human blood.

11. The method of claim 1, wherein the predetermined period of time ranges from 1 minute to 5 minutes.

12. The method of claim 1, wherein the treated platelet rich plasma has a range of 0.5 x 10 when analyzed immediately after the predetermined period of time ⁶ /mL to 2.0X10 ⁶ Amount of stem cells per mL.

13. A method of producing a composition that reduces the physiological age of a patient when administered to the patient, the method comprising:

Proliferating stem cells of the patient, comprising:

adding normal human blood to the plurality of tubes;

centrifuging the plurality of tubes at a predetermined g-force for 10 minutes to produce platelet rich plasma;

shaking the plurality of tubes;

aliquoting said produced platelet rich plasma into sterile tubes;

shaking the platelet rich plasma in the sterile tube;

treating the platelet rich plasma with a modulated pulse of laser light having a predetermined wavelength for a predetermined period of time; and

shaking the treated platelet rich plasma.

14. The method of claim 13, wherein the plurality of tubes ranges from 3 tubes to 12 tubes, and any increments therein.

15. The method of claim 13, wherein said treatment of said platelet rich plasma is performed under minimal background white light conditions.

16. The method of claim 13, wherein the predetermined wavelength range is 300nm to 1000nm.

17. The method of claim 13, wherein the predetermined wavelength is 670nm.

18. The method of claim 13, wherein the predetermined period of time ranges from 1 minute to 5 minutes.

19. The method of claim 13, wherein the treated platelet rich plasma has a range of 0.5 x 10 when analyzed immediately after the predetermined period of time ⁶ /mL to 2.0X10 ⁶ Amount of stem cells per mL.

20. The method of claim 13, wherein the treated platelet rich plasma exhibits a 2.5-fold increase in stem cells compared to the average of a first control sample and a second control sample, wherein the first control sample comprises the platelet rich plasma treated with white flashlight light for a predetermined period of time, and wherein the second control sample comprises the platelet rich plasma not treated with any light.

21. The method of claim 13, wherein the modulating eliminates a center wavelength band of the laser such that the remaining upper and lower wavelength bands create beat patterns of sparse nodes.