What is exosome | Smart Clinic Ginza

High Concentration Factor 9
in our clinic

About stem cell culture supernatant

Our cells are damaged every day due to injuries and aging.
What repairs these cells are "stem cells" that differentiate themselves into various cells and promote regeneration.
"Stem cell culture supernatant" is the clear liquid secreted from the upper layer of the precipitate when stem cells are cultured.
It contains thousands of growth factors (physiologically active substances) such as exosomes and cytokines, which can be expected to produce the same effect as stem cells.

Through the action of exosomes, effects such as cell activation, promotion of tissue regeneration, and improvement of blood circulation can be expected.
It is also attracting attention in the field of anti-aging. Among the stem cells derived from various human tissues such as fat, bone marrow, and umbilical cord, the pulp stem cells of milk teeth contain the most growth factors.

Reasons to use dental pulp stem cell culture supernatant (exosomes)

Advantages offered by the growth environment of dental pulp

Dental pulp is surrounded by hard teeth and is therefore placed in a harsh environment with a very low blood supply compared to other stem cell culture supernatants. It is because of being placed in such an environment that it grows into strong and undamaged cells.
The active capacity of pulp grown in such an environment is very high, with a consequent increase in cell division and proliferation. The greater the proliferation, the greater the variety and concentration of exosomes and cytokines.

Discoverer of stem cell culture supernatant

Dr. Minoru Ueda

Professor Emeritus, Nagoya University School of Medicine
Advisor, Japan Society of Regenerative Medicine

Doctor of Medicine

Specialized field: regenerative medicine and maxillofacial surgery

1949: Born in Osaka
1982: Postgraduate study at Nagoya University School of Medicine
Joining the Oral Surgery Department of Nagoya University (as a lecturer and assistant professor in the same department)
1990: Studied at the University of Gothenburg, Sweden and the University of Zurich, Switzerland
1994: Assumed the position of Chair Professor of Maxillofacial Surgery at Nagoya University School of Medicine
2003〜2008: Adjunct Professor, Graduate School of Medical Sciences, University of Tokyo
2004: Received the President's Award of the Japan Academic Conference
2011: Visiting Professor, University of Bergen, Norway
2015: Appointed Honorary Professor of Nagoya University Faculty of Medicine
2016: Honored with the Johnson&Johnson Award of the Japanese Society for Regenerative Medicine, Advisor of the Japanese Society for Regenerative Medicine Honorary member of the Japanese Society for Inflammatory Regenerative Medicine, in charge of research and clinical guidance on regenerative medicine.

Published more than 600 papers on cells, with more than 300 cases of regenerative medicine applied to skin and bone.
Evaluation committee member of the Japan Agency for Medical Research and Development (AMED), Chairman of the Institute of Regenerative Medicine, Inc.

High Concentration Factor 9 in our clinic

In collaboration with a GMP-compliant cell processing center in Japan, we have independently developed exosomes (stem cell culture supernatant), named them "High Concentration Factor 9," and used them in the form of drips.
Our "High Concentration Factor 9" is a high-quality liquid preparation containing more than 5,000 types of cytokine proteins and a high concentration of exosomes.

4 main points of High Concentration Factor 9

Infinite proliferation is possible, and cell supply is stable and continuous .

It is reported that the content of growth factors and cytokines is about 30 ~ 50 times that of primary cells.

It is reported that it is effective on plural cell types. and may be useful for regeneration of various tissues and organs.

The raw materials produced in class100 (Grade A), class10,000 (Grade B) cleanrooms have passed quality specification tests conducted by third-party organizations under stringent standards.

High cellular activity in combination with cytokine W

In the stock solution of stem cell culture supernatant, impurities such as lactic acid and ammonia released from the cells are also present. Our "High Concentration Factor 9" removes these impurities through a refining process.
A cell activity test was conducted by varying the concentration of three solutions: stem cell culture supernatant stock solution, our High Concentration Factor 9, and exosome-only extract, and found that the High Concentration Factor 9 showed higher cell activity than the exosome-only extract.

Content of the cell activity test and its results

Experimental content

Stock solutions for stem cell culture supernatant
High Concentration Factor 9
Extracts of exosomes only

Experiments to detect the degree of cellular activity when added to cells by varying the concentration of the three solutions.

Experimental results

The stem cell culture supernatant stock solution of 1 and the exosome-only extract of 3 show a drastic decrease in activity at high concentrations, which can be confirmed by the graphs on the right (red arrows).
This should be due to the influence of impurities. In addition, it can be confirmed that the degree of activity of the exosome-only extract is also low. Our "High Concentration Factor 9" has been proven in experiments to increase cellular activity even at high concentrations.

Results of the cell activity experiment

Stem cell culture supernatant and exosome droplets that do not show the results of the test may not have been refined by removing impurities and concentrating, or the cell activity test may not have been performed.

Reliability

We use pulp stem cells from a defined source.

Our "High Concentration Factor 9" collects deciduous teeth from Japanese children and cultures pulp stem cells. It is reviewed by the Ethics Committee, which is indispensable for research in the Faculty of Life Sciences and Medicine that targets human beings, and tooth extraction and pulp tissue collection are performed in an optimal procedure.

In addition, it was confirmed that dental pulp stem cells were positive for CD73, CD90, and CD105 and negative for CD34 and CD45 among the cell surface markers that indicate MSCs.
This proved to be MSCs with the ability to differentiate into various functional cells.

Results of surface marker test data

CD73:Positive

CD90:Positive

CD105:Positive

CD34:Negative

CD45:Negative

We do not use pulp stem cells of unknown origin or stem cells that cannot be identified as mesenchymal stem cells in our High Concentration Factor 9.

It is strictly produced in a GMP-compliant cell processing center.

Our "High Concentration Factor 9" is manufactured in a cell processing center that complies with the strict GMP (Good Manufacturing Practice) standards. In addition to GMP, it also complies with the following provincial ordinances and standard guidelines.

Compliance with Provincial Orders

GMPDrug Manufacturing Management and Quality Control Standards
GCTPProduction management and quality management standards for regenerative medicine and other products
GQPStandards defining product quality management methods for GQP drugs, quasi-drugs, cosmetics, and regenerative medicine, etc.
Laws to ensure the safety of regenerative medicine, etc.

Guidelines for Compliance with Specifications and Standards

ISO(International Organization for Standardization)
An international organization that defines various quality standards.
ICH(International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use) (International Conference on Drug Control Harmonization)
An international organization that promotes guidelines for pharmaceutical standards worldwide
MISEV2018MISEV2018 is a guideline for extracellular vesicle (EV) research standards published by the International Society for Extracellular Vesicles (ISEV) in 2018.

We do not use stem cell culture supernatants and exosomes produced in facilities with vague standards of production control, quality control, and hygiene control for our High Concentration Factor 9.

Examination of cells upon receipt and product shipment

The Cell Processing Center conducts safety tests at the time of receipt of "High Concentration Factor 9" basic cells and at the time of shipment of the product. Only cells that have passed these tests can be used for "High Concentration Factor 9" at our clinic.

Inspection at the time of cell acceptance

In accordance with the Biological Material Standards of the Ministry of Health, Labor and Welfare (established on February 28, 2007, Ministry of Health, Labor and Welfare Notification No. 37), the following safety tests are conducted.

Sterility test
Endotoxin test/Mycoplasma test
Virus negative tests (HBs antigen-antibody, HBc antibody, HTLV-I antibody (CLIA), HIV antigen-antibody, syphilis (PRP/TPHA))

Inspection of supernatant upon shipment from the factory

Sterility test
Endotoxin test/Mycoplasma test
Cell-free examination

Total quality management through 4 major safety tests

Our "High Concentration Factor 9" was confirmed to be negative in all of the following tests.

Tests for human viruses (HBV/HCV/HIV/HTLV-I/HIV antigen/antibody/RPR/TPHA) recommended in the Guidelines for Quality and Safety of Cellular Processed Drugs. Nucleic acids were extracted from cells collected by centrifugation of test samples while performing DNA/RNA recovery procedures using NucleoSpin RNA, and the presence of viruses was determined by reverse transcription/real-time PCR using specific primer/probe combinations for each viral sequence.

Aerobic bacteria such as molds that proliferate in culture and anaerobic bacteria that do not require oxygen are measured. Using a sterility test device bacterial alarm system, a test sample is inoculated in a test medium and incubated at 32.7°C for 7 days, and the sterility of the bacteria is determined from their proliferation. In addition, as an obstruction confirmation test, test medium is inoculated with test samples and various standard bacteria and incubated in the same manner as the test samples. The standard bacteria also grew, and it was thus determined that there was no hindrance.

Mycoplasmas are the smallest microorganisms with the ability to self-proliferate, biologically classified as bacteria, and widely known for their ability to cause pneumonia, among other things. It is difficult to observe the bacteria because they are too small and have no cell wall. Therefore, the presence of Mycoplasma was determined by real-time PCR using the MycoSEQMycoplasma Detection Kit, which extracts DNA from the sample and the sample plus positive control DNA, using Mycoplasma-specific primers.

Endotoxin is a pyrogen (febrile substance) that has biological activities such as febrile action and lethal toxicity. Samples are added to the Endosafe-PTS kit JP and measured using an endotoxin measuring device. The recovery rate of the addition of the endotoxin test control and the endotoxin test concentration of the samples are measured using the endotoxin control added to the kit as an indicator.

Extremely pure exosome supernatant liquid “High Concentration Factor 9”

Our "High Concentration Factor 9" is fine exosome supernatant of high purity with unlimited removal of impurities such as ammonia and lactic acid in the concentration process.

Removal of impurities is time consuming and specialized knowledge. Untreated stock solution has a high concentration of impurities, and poor quality stock solution may cause side effects if it is returned to the body by drip, so it is not used in our clinic.