KR20230017722A - Composition for wound healing or accelerating wound healing comprising exosomes derived from cord blood progenitor cell - Google Patents
Composition for wound healing or accelerating wound healing comprising exosomes derived from cord blood progenitor cell Download PDFInfo
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- KR20230017722A KR20230017722A KR1020220081501A KR20220081501A KR20230017722A KR 20230017722 A KR20230017722 A KR 20230017722A KR 1020220081501 A KR1020220081501 A KR 1020220081501A KR 20220081501 A KR20220081501 A KR 20220081501A KR 20230017722 A KR20230017722 A KR 20230017722A
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Abstract
Description
본 발명은 제대혈간세포(cord blood progenitor cell) 유래 엑소좀(exosome)을 포함하는 창상 치료 또는 창상 치료 촉진용 조성물에 관한 것이다.The present invention relates to a composition for wound healing or promoting wound healing comprising exosomes derived from cord blood progenitor cells.
창상피복재는 습윤드레싱으로도 불리며 창상 부위의 오염 방지 및 피부보호, 삼출액의 흡수, 출혈 또는 체액손실 방지 등의 목적으로 만들어지는 의료기기이다. Wound dressings, also called wet dressings, are medical devices made for the purpose of preventing contamination of wounds, protecting skin, absorbing exudate, and preventing bleeding or loss of body fluids.
창상은 피부에 발생한 상처로서 상처부위의 여러 면역세포에 의해 분비되는 다양한 사이토카인에 의해 염증반응이 발생한 후 피부의 증식 및 성숙단계를 거쳐 치유가 된다. 창상 치료에 있어 가장 이상적인 피복재는 창상부위가 외부환경에 노출되는 것을 차단하여 감염을 예방하고, 염증반응을 억제하고, 상처치유를 촉진하는 것이다. A wound is a wound that occurs on the skin, and after an inflammatory reaction is generated by various cytokines secreted by various immune cells at the wound site, it is healed through the proliferation and maturation stages of the skin. The most ideal covering material for wound treatment prevents infection by blocking exposure of the wound to the external environment, inhibits inflammatory reactions, and promotes wound healing.
그러나 지금까지의 창상피복재는 콜라겐, 히알루론산 등 다양한 생체재료를 이용하여 삼출물을 흡수하고 보호하는 물리적인 역할을 할 뿐 창상조직에서 발생하는 염증 및 상처치유에 대한 적극적인 역할은 수행할 수 없었다. 또한, 종래 하이드로 화이버형 등 창상피복재는 삼출물 흡수, 미생물 감염 예방, 피부재생 등 목적을 위주로 개발되어, 면역세포 조절을 통한 염증반응 억제 효과가 미미하다는 단점을 갖는다.However, so far, wound dressing materials have only played a physical role of absorbing and protecting exudate using various biomaterials such as collagen and hyaluronic acid, but could not play an active role in inflammation and wound healing occurring in wound tissue. In addition, conventional wound dressing materials such as hydrofiber type have been developed mainly for purposes such as absorbing exudate, preventing microbial infection, and regenerating the skin, and have a disadvantage in that the effect of suppressing inflammatory reactions by regulating immune cells is insignificant.
따라서, 창상으로 인해 손상된 피부조직을 재생하고 흉터를 최소화하기 위해서는 기존의 창상피복재의 효능에 더해 창상부위의 염증반응을 억제하고 상처치유를 촉진할 수 있는 새로운 창상피복재의 개발이 필요하다.Therefore, in order to regenerate damaged skin tissue and minimize scarring, it is necessary to develop a new wound dressing material capable of suppressing the inflammatory response of the wound site and promoting wound healing in addition to the efficacy of conventional wound dressing materials.
종래의 창상피복재에 대한 문제점을 해결하고자, 중간엽줄기세포(mesenchymal stem cell, MSC)를 활용한 창상피복재를 개발하고자 하는 노력이 진행되고 있으며, 특히, 중간엽줄기세포 분비인자인 엑소좀 등을 이용한 창상피복재 개발도 시도되고 있다.In order to solve the problems of conventional wound dressings, efforts are being made to develop wound dressings using mesenchymal stem cells (MSC). The development of a wound covering material using this method is also being attempted.
이와 관련하여, 중간엽줄기세포에서 유도된 엑소좀들이 창상치유와 같은 조직 재생을 유도할 수 있다는 것을 입증하는 많은 문헌들이 있으나, 제대혈 단핵세포(cord blood mononuclear cell)로부터 수득한 제대혈간세포(cord blood progenitor cell) 유래 엑소좀이 기존의 중간엽줄기세포 유래 엑소좀들에 비해서 창상치유에 우수하다는 것은 전혀 알려진 바가 없다.In this regard, there are many literatures proving that exosomes derived from mesenchymal stem cells can induce tissue regeneration such as wound healing, but cord blood stem cells obtained from cord blood mononuclear cells It is not known at all that progenitor cell-derived exosomes are superior to conventional mesenchymal stem cell-derived exosomes in wound healing.
본 발명자들은 제대혈간세포 유래 엑소좀(exosome)에서 우수한 염증반응 완화 효과, 상처 치유, 세포 이동 및 면역세포 침윤현상의 회복효과를 확인하여, 상처 부위의 심출물 및 가려움증을 최소화할 수 있는 우수한 창상 치료 용도를 확인함으로써 본 발명을 완성하게 되었다. The present inventors confirmed the excellent inflammatory reaction mitigating effect, wound healing, cell migration and recovery effect of immune cell infiltration in exosomes derived from cord blood stem cells, and excellent wound treatment that can minimize exudation and itching in the wound area The present invention was completed by confirming the use.
따라서, 본 발명의 목적은 제대혈간세포 유래 엑소좀을 유효성분으로 포함하는 창상 치료 또는 창상 치료 촉진용 약학적 조성물을 제공하는 것에 있다. Accordingly, an object of the present invention is to provide a pharmaceutical composition for wound healing or promoting wound healing comprising exosomes derived from cord blood stem cells as an active ingredient.
본 발명의 다른 목적은 본 발명의 약학적 조성물을 포함하는 창상 치료 또는 창상 치료 촉진용 약학 제제를 제공하는 것에 있다.Another object of the present invention is to provide a pharmaceutical preparation for wound healing or promoting wound healing comprising the pharmaceutical composition of the present invention.
본 발명의 다른 목적은 본 발명의 약학적 조성물을 포함하는 창상 개선용 의약외품 제제를 제공하는 것에 있다.Another object of the present invention is to provide a quasi-drug preparation for wound healing comprising the pharmaceutical composition of the present invention.
본 발명의 다른 목적은 본 발명의 약학적 조성물을 포함하는 창상 치료 또는 창상 치료 촉진용 의료기기를 제공하는 것에 있다.Another object of the present invention is to provide a medical device for wound healing or promoting wound healing comprising the pharmaceutical composition of the present invention.
본 발명의 다른 목적은 본 발명의 약학적 조성물의 제조방법을 제공하는 것에 있다.Another object of the present invention is to provide a method for preparing the pharmaceutical composition of the present invention.
본 발명이 해결하고자 하는 과제들은 이상에서 언급된 과제로 제한되지 않으며, 언급되지 않은 또 다른 과제들은 아래의 기재로부터 통상의 기술자에게 명확하게 이해될 수 있을 것이다.The problems to be solved by the present invention are not limited to the problems mentioned above, and other problems not mentioned will be clearly understood by those skilled in the art from the description below.
상술한 기술적 과제를 해결하기 위해, 본 발명의 일 양태에 따르면, 제대혈간세포 유래 엑소좀을 유효성분으로 포함하는 창상 치료 또는 창상 치료 촉진용 약학적 조성물을 제공한다.In order to solve the above technical problems, according to one aspect of the present invention, a pharmaceutical composition for wound healing or promoting wound healing comprising exosomes derived from cord blood stem cells as an active ingredient is provided.
본 발명에서 용어 “제대혈”은 태반과 태아를 연결하는 제대정맥으로부터 채취된 혈액을 의미한다. 제대혈은 출산 시 자연적으로 발생하는 부산물로서, 여러 차례의 수술을 필요로 하는 골수 등의 일반 간엽조직보다 훨씬 채취가 용이하고, 골수 이식에 비해 제대혈 보관산업이 활성화되어 인프라가 구축되어 있으므로 공여자를 구하는 것이 용이하다. 이와 더불어, 제대혈 유래 세포는 조직이나 장기 이식에서 거부반응을 일으키는 가장 중요한 원인인 조직적합항원 HLA-DR(class II)이 발현되지 않는 세포이므로, 기존의 이식수술시 문제가 되었던 거부반응등의 면역반응을 유발하지 않거나 최소화할 수 있어, 자가유래 제대혈은 물론 타가유래 제대혈 또한 사용할 수 있다. In the present invention, the term "umbilical cord blood" refers to blood collected from the umbilical vein connecting the placenta and the fetus. Cord blood is a naturally occurring by-product during childbirth, and is much easier to collect than normal mesenchymal tissue such as bone marrow, which requires multiple operations. it is easy In addition, cord blood-derived cells are cells that do not express the histocompatibility antigen HLA-DR (class II), which is the most important cause of rejection in tissue or organ transplantation. Since the reaction is not induced or minimized, not only autologous umbilical cord blood but also other-derived umbilical cord blood can be used.
본 발명에서 용어 “제대혈간세포”는 상기 제대혈 유래 전구 세포 (progenitor cell)를 의미한다. In the present invention, the term “umbilical cord blood stem cells” refers to the cord blood-derived progenitor cells.
본 발명에서 용어 “엑소좀(exosome)”은 세포막의 구조와 동일한 이중인지질막으로 이루어진 소낭체를 의미한다. 생리학적 조건에서 모든 생물체의 세포는 엑소좀를 분비한다. 미세소포(microvesicals), 세포자멸사 소체(apoptotic bodies)등 기타 세포외 소낭체들의 크기도 엑소좀과 비슷하지만 생물발생 경로가 다르다. 엑소좀의 가장 큰 역할은 카고(cargo) 즉 물질운반체로서 단백질, 지질, mRNA, microRNA, long non-coding RNA 등 핵산정보를 운반한다. 생리학적 조건에서 엑소좀은 지속적으로 세포와 세포사이를 오가면서 이런 정보를 전달하여 세포들의 교류에 중요한 역할을 한다. In the present invention, the term “exosome” refers to a vesicle composed of a double-phospholipid membrane identical to the structure of a cell membrane. Under physiological conditions, cells of all organisms secrete exosomes. Other extracellular vesicles, such as microvesicles and apoptotic bodies, are similar in size to exosomes, but have different biogenesis pathways. The most important role of exosomes is cargo, that is, a material transporter, which carries nucleic acid information such as proteins, lipids, mRNAs, microRNAs, and long non-coding RNAs. Under physiological conditions, exosomes play an important role in cell-to-cell communication by continuously transferring information between cells.
본 발명에서 용어 “창상”은 표피, 진피 또는 피하조직의 결손에 의한 것임을 특징으로 한다.In the present invention, the term “wound” is characterized by defects in the epidermis, dermis, or subcutaneous tissue.
본 발명에서 용어 “치료”는 (i) 질환, 질병 또는 증상의 발전의 억제, (ⅱ) 질환, 질병 또는 증상의 경감, 또는 (ⅲ) 질환, 질병 또는 증상의 제거를 의미한다. As used herein, the term “treatment” refers to (i) inhibition of the development of a disease, disorder or condition, (ii) alleviation of a disease, disorder or condition, or (iii) elimination of a disease, disorder or condition.
본 발명의 “창상 치료”는 본 발명의 약학적 조성물의 투여로 인해 창상부위에서의 항염증 효과, 혈관손상에 의한 면역세포 침윤의 회복, 상처 치유 및/또는 세포이동 효과에 의한 것임을 특징으로 한다. The "wound treatment" of the present invention is characterized by anti-inflammatory effect at the wound site, recovery of immune cell infiltration due to blood vessel damage, wound healing and/or cell migration effect due to administration of the pharmaceutical composition of the present invention. .
본 발명의 일 구현예에 따르면, 상기 제대혈간세포는 표면마커 중에서 CD24, CD117 및 SSEA-1에 대하여 음성의 면역학적 특성을 나타내고, CD31, CD45RA, CD105, CD146 및/또는 TRA-1-60에 대하여 양성의 면역학적 특성을 나타내는 것을 특징으로 한다.According to one embodiment of the present invention, the cord blood stem cells exhibit negative immunological characteristics for CD24, CD117 and SSEA-1 among surface markers, and for CD31, CD45RA, CD105, CD146 and/or TRA-1-60 It is characterized by exhibiting positive immunological properties.
본 발명의 일 구현예에 따르면, 상기 엑소좀은 표면마커 중에서 CD9, CD63, CD81 외에 CD44, CD53 및/또는 CD98을 더 포함하는 것을 특징으로 한다.According to one embodiment of the present invention, the exosome is characterized in that it further contains CD44, CD53 and/or CD98 in addition to CD9, CD63 and CD81 among surface markers.
본 발명의 실시예에서 상기 엑소좀은 (a) 단핵세포(mononuclear cell)를 냉동한 후 해동하는 한랭쇼크 방법으로 제대혈간세포를 획득하는 단계; (b) 제1배지에서 상기 제대혈간세포를 배양하여 세포배양 플라스크에 부착시키는 단계; (c) (b)의 결과물을 제2배지에서 1주 내지 5주 동안 배양하는 단계; (d) (c)의 결과물이 분비하는 엑소좀을 수집하는 단계; 및 (e) 상기 수집된 엑소좀을 분리하는 단계를 통해 생산하였다. In an embodiment of the present invention, the exosomes are prepared by (a) obtaining umbilical cord blood stem cells by a cold shock method of freezing mononuclear cells and then thawing them; (b) culturing the cord blood stem cells in a first medium and attaching them to a cell culture flask; (c) culturing the product of (b) in a second medium for 1 to 5 weeks; (d) collecting the exosomes secreted by the product of (c); and (e) isolating the collected exosomes.
상기 제1배지는 글루타민(L-glutamine) 또는 L-글루타민의 dipeptide 형태(L- alanyl-L-glutamine dipeptide), 우태아혈청(FBS), 피루브산 나트륨(Sodium pyruvate), 불필수아미노산(Non-essential amino acids), 트랜스페린(Transferrin), 소듐 셀레나이트(Sodium selenite) 및 인슐린(Insulin)으로 이루어진 군에서 선택된 하나 이상이 추가된 둘베코변형 이글 배지(DMEM: Dulbecco’2 Modified Eagle Medium), Advanced DMEM, 최소 필수 배지(MEM: Minimal Essential Medium), IMDM(Iscove’s Modified Dulbecco’s Medium) 또는 DMEM/F12 배지를 포함한다. The first medium is glutamine (L-glutamine) or L-glutamine dipeptide form (L-alanyl-L-glutamine dipeptide), fetal bovine serum (FBS), sodium pyruvate (Sodium pyruvate), non-essential amino acid amino acids), Dulbecco'2 Modified Eagle Medium (DMEM) with one or more selected from the group consisting of Transferrin, Sodium selenite and Insulin added, Advanced DMEM, Includes Minimal Essential Medium (MEM), Iscove's Modified Dulbecco's Medium (IMDM) or DMEM/F12 medium.
상기 제2배지는 Glutathione monosodium, 비타민 B12, 모노소듐 하이포크산틴(Monosodium hypoxanthine), 티미딘(Thymidine), 글루타민(L-glutamine) 및 L-글루타민의 디펩타이드 형태(L-alanyl-L-glutamine dipeptide), 녹아웃 혈청 대체물(Knockout serum replacement)으로 이루어진 군에서 선택된 하나 이상이 추가된 둘베코변형 이글 배지(DMEM: Dulbecco’2 Modified Eagle Medium), 최소 필수 배지(MEM: Minimal Essential Medium), IMDM(Iscove’s Modified Dulbecco’s Medium), DMEM/F12(Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12) 또는 DMEM/F-12 Glutamax supplement 배지를 포함한다. The second medium is glutathione monosodium, vitamin B12, monosodium hypoxanthine, thymidine, glutamine (L-glutamine), and a dipeptide form of L-glutamine (L-alanyl-L-glutamine dipeptide) , Dulbecco'2 Modified Eagle Medium (DMEM), Minimal Essential Medium (MEM), and IMDM (Iscove's Modified) with one or more added selected from the group consisting of knockout serum replacement. Dulbecco's Medium), DMEM/F12 (Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12) or DMEM/F-12 Glutamax supplement medium.
본 발명의 약학적 조성물에 포함되는 약학적으로 허용되는 담체는 제제시에 통상적으로 이용되는 것으로서, 탄수화물류 화합물 (예: 락토스, 아밀로스, 덱스트로스, 수크로스, 솔비톨, 만니톨, 전분, 셀룰로스, 등), 아카시아 고무, 인산 칼슘, 알기네이트, 젤라틴, 규산 칼슘, 미세결정성 셀룰로스, 폴리비닐피롤리돈, 셀룰로스, 물, 시럽, 염 용액, 알코올, 아라비아 고무, 식물성 기름 (예: 옥수수 기름, 목화 종자유, 두유, 올리브유, 코코넛유), 폴리에틸렌 글리콜, 메틸 셀룰로스, 메틸히드록시 벤조에이트, 프로필히드록시 벤조에이트, 활석, 스테아르산 마그네슘 및 미네랄 오일 등을 포함하나, 이에 한정되는 것은 아니다. The pharmaceutically acceptable carrier included in the pharmaceutical composition of the present invention is one commonly used in formulation, and includes carbohydrate compounds (e.g., lactose, amylose, dextrose, sucrose, sorbitol, mannitol, starch, cellulose, etc.) ), gum acacia, calcium phosphate, alginates, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, salt solutions, alcohol, gum arabic, vegetable oils (e.g. corn oil, cotton) seed oil, soy milk, olive oil, coconut oil), polyethylene glycol, methyl cellulose, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate and mineral oil, and the like, but are not limited thereto.
본 발명의 약학적 조성물은 상기 성분들 이외에 윤활제, 습윤제, 감미제, 향미제, 유화제, 현탁제, 보존제 등을 추가로 포함할 수 있다. 적합한 약제학적으로 허용되는 담체 및 제제는 Remington's Pharmaceutical Sciences (19th ed., 1995)에 상세히 기재되어 있다. The pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, and the like in addition to the above components. Suitable pharmaceutically acceptable carriers and agents are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).
본 발명의 약학적 조성물의 적합한 투여량은 제제화 방법, 투여 방식, 환자의 연령, 체중, 성, 병적 상태, 음식, 투여 시간, 투여 경로, 배설 속도 및 반응 감응성과 같은 요인들에 의해 다양하게 처방될 수 있다. A suitable dosage of the pharmaceutical composition of the present invention is variously prescribed depending on factors such as formulation method, administration method, patient's age, weight, sex, pathological condition, food, administration time, administration route, excretion rate and response sensitivity. It can be.
본 발명의 약학적 조성물은 당해 발명이 속하는 기술분야에서 통상의 지식을 가진 자가 용이하게 실시할 수 있는 방법에 따라, 약학적으로 허용되는 담체 및/또는 부형제를 이용하여 제제화함으로써 단위 용량 형태로 제조되거나 또는 다용량 용기내에 내입시켜 제조될 수 있다. 이때 제형은 오일 또는 수성 매질중의 용액, 현탁액 또는 유화액 형태이거나 엑스제, 분말제, 과립제, 정제 또는 캅셀제 형태일 수도 있으며, 분산제 또는 안정화제를 추가적으로 포함할 수 있다.The pharmaceutical composition of the present invention is prepared in unit dosage form by formulation using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily performed by those skilled in the art. or it may be prepared by incorporating into a multi-dose container. In this case, the formulation may be in the form of a solution, suspension or emulsion in an oil or aqueous medium, or may be in the form of an extract, powder, granule, tablet or capsule, and may additionally contain a dispersing agent or stabilizer.
본 발명의 일 구현예에 따르면, 상기 조성물은 정제수, 폴리올, 히알루론산 및 방부제으로 이루어진 그룹에서 선택된 적어도 하나의 보조성분을 더 포함할 수 있다.According to one embodiment of the present invention, the composition may further include at least one auxiliary component selected from the group consisting of purified water, polyol, hyaluronic acid, and preservatives.
상기 폴리올은 프로판디올(Propanediol), 글리세린(Glycerin), 부틸렌글라이콜(Butylene Glycol), 프로필렌글리콜(Propylene Glycol), 디프로필렌글리콜(Dipropylene Glycol), 펜틸렌글라이콜(Pentylene Glycol), 헥실렌글라이콜(Hexylene Glycol), 폴리에틸렌글라이콜(Polyethylene Glycol), 만니톨(Mannitol) 및 솔비톨(Sorbitol) 로 이루어진 그룹에서 선택된 적어도 하나일 수 있다.The polyol is Propanediol, Glycerin, Butylene Glycol, Propylene Glycol, Dipropylene Glycol, Pentylene Glycol, Hexylene Glycol It may be at least one selected from the group consisting of Hexylene Glycol, Polyethylene Glycol, Mannitol, and Sorbitol.
상기 히알루론산은 음이온성 다당류, 구체적으로는 리코사미노글리칸이다. 히알루론산은 히알루론산 및 임의의 이의 히알루로네이트 염을 지칭하며, 이러한 염에는 소듐 하이알루로네이트, 포타슘 하이알루로네이트, 마그네슘 하이알루로네이트 및 칼슘 하이알루로네이트로 이루어진 그룹에서 선택된 적어도 하나일 수 있다. The hyaluronic acid is an anionic polysaccharide, specifically a lycosaminoglycan. Hyaluronic acid refers to hyaluronic acid and any of its hyaluronate salts, including at least one selected from the group consisting of sodium hyaluronate, potassium hyaluronate, magnesium hyaluronate and calcium hyaluronate. can be
상기 방부제는 글리세릴카프릴레이트 (glyceryl caprylate, GMCY), 4-하이드록시아세토페논(4-hydroxyacetophenone), 헥산디올(1,2-hexandiol), 및 에틸헥실글리세린(ethylhexyl glycerin)으로 이루어진 그룹에서 선택된 적어도 하나일 수 있다. The preservative is at least one selected from the group consisting of glyceryl caprylate (GMCY), 4-hydroxyacetophenone, 1,2-hexandiol, and ethylhexyl glycerin. can be one
본 발명의 실시예에서 상기 조성물은 정제수, 펜틸렌글라이콜(Pentylene glycol), 소듐하이알루로네이트(Sodium hyaluronate), 소듐클로라이드(Sodium chloride), 다이소듐포스페이트(Disodium phosphate), 만니톨(Mannitol), 소퓸포스페이트(Sodium phosphate) 및 하이드록시아세토페논(Hydroxyacetophenone)으로 이루어진 그룹에서 선택된 적어도 하나의 보조성분을 더 포함한다. In an embodiment of the present invention, the composition is purified water, pentylene glycol, sodium hyaluronate, sodium chloride, disodium phosphate, mannitol, It further includes at least one auxiliary component selected from the group consisting of sodium phosphate and hydroxyacetophenone.
본 발명의 다른 양태에 따르면, 본 발명은 상기 약학적 조성물을 포함하는 창상 치료 또는 창상 치료 촉진용 약학 제제를 제공한다.According to another aspect of the present invention, the present invention provides a pharmaceutical preparation for wound healing or promoting wound healing comprising the above pharmaceutical composition.
본 발명의 일 구현예에 따르면, 상기 제제는 주사제형, 주입제형, 분무제형, 액상제형 또는 패취제형인 것을 특징으로 한다.According to one embodiment of the present invention, the preparation is characterized in that it is in the form of an injection, injection, spray, liquid or patch.
상기 제제는 약학적으로 허용가능한 담체를 더 포함하는 것을 특징으로 한다.The formulation is characterized in that it further comprises a pharmaceutically acceptable carrier.
본 발명의 다른 양태에 따르면, 본 발명은 상기 약학적 조성물을 포함하는 창상 개선용 의약외품 제제를 제공한다.According to another aspect of the present invention, the present invention provides a quasi-drug preparation for wound healing comprising the pharmaceutical composition.
본 발명의 일 구현예에 따르면, 상기 의약외품 제제는 액제, 연고제, 크림제, 스프레이제, 패취제, 겔제 및 에어로졸제로 이루어지는 군으로부터 선택되는 피부 외용제 형태일 수 있으며, 이에 한정되는 것은 아니다.According to one embodiment of the present invention, the quasi-drug preparation may be in the form of an external preparation for skin selected from the group consisting of solutions, ointments, creams, sprays, patches, gels, and aerosols, but is not limited thereto.
본 발명의 다른 양태에 따르면, 본 발명은 상기 약학적 조성물을 포함하는 창상 치료 또는 창상 치료 촉진용 의료기기를 제공한다.According to another aspect of the present invention, the present invention provides a medical device for wound healing or promoting wound healing comprising the pharmaceutical composition.
본 발명의 일 구현예에 따르면, 상기 의료기기는 창상 피복재 또는 지혈용 기재일 수 있으며, 이에 한정되는 것은 아니다.According to one embodiment of the present invention, the medical device may be a wound covering material or a substrate for hemostasis, but is not limited thereto.
본 발명의 조성물은 조직 재생 효과를 갖는 필러로 활용될 수 있다.The composition of the present invention can be used as a filler having a tissue regeneration effect.
본 발명의 조성물은 기능성 화장품 제형으로 활용될 수 있으며, 특히, 기능성 화장품 제형은 당업계에서 통상적으로 제조되는 어떠한 제형으로도 제조될 수 있다. 예를 들어 패취, 마스크팩, 마스크시트, 유연화장수, 영양화장수, 수렴화장수, 영양크림, 마사지크림, 아이크림, 클렌징크림, 에센스, 아이에센스, 클렌징로션, 클렌징폼, 클렌징워터, 선스크린, 립스틱, 비누, 샴푸, 계면활성제-함유 클렌징, 입욕제, 바디로션, 바디크림, 바디오일, 바디에센스, 바디세정제, 염모제 및 헤어토닉 등으로 제형화할 수 있으나 이에 한정되는 것은 아니다.The composition of the present invention can be used as a functional cosmetic formulation, and in particular, the functional cosmetic formulation can be prepared in any formulation conventionally prepared in the art. For example, patches, mask packs, mask sheets, softening lotion, nourishing lotion, astringent lotion, nourishing cream, massage cream, eye cream, cleansing cream, essence, eye essence, cleansing lotion, cleansing foam, cleansing water, sunscreen, lipstick , soap, shampoo, surfactant-containing cleansing, bath additives, body lotion, body cream, body oil, body essence, body cleanser, hair dye and hair tonic, etc., but are not limited thereto.
상기 의약외품 제제, 피부 외용제 및/또는 기능성 화장품의 조성물은 의약외품 제제, 피부 외용제 및/또는 기능성 화장품의 조성물에 통상적으로 이용되는 성분들을 포함하며, 예컨대 항산화제, 안정화제, 용해화제, 비타민, 안료 및 향료와 같은 통상적인 보조제 그리고 담체를 포함할 수 있다. The composition of the quasi-drug preparation, skin external preparation, and/or functional cosmetics includes ingredients commonly used in the quasi-drug preparation, skin external preparation, and/or functional cosmetic composition, such as antioxidants, stabilizers, solubilizers, vitamins, pigments and Usual adjuvants such as flavoring agents and carriers may be included.
본 발명의 다른 양태에 따르면, 본 발명은 다음의 단계를 포함하는, 약학적 조성물의 제조방법을 제공한다: (a) 제대혈간세포 배양액을 필터에 통과시켜 세포 잔해물, 노폐물, 거대 입자 중 적어도 어느 하나를 여과하는 단계; (b) 상기 여과된 배양액을 멸균 필터로 멸균하는 단계; (c) 상기 멸균된 배양액을 접선흐름여과방법을 이용하여 농축과 정용여과과정을 통해 엑소좀을 분리하는 단계; (d) 상기 분리된 엑소좀에 안정화 버퍼를 추가하여 다시 시작 부피가 되도록 희석하는 단계; 및 (e) 상기 희석된 엑소좀을 액체 교환 시스템으로 멸균 필터에 통과시켜 멸균한 후 밀봉된 저장용기에 저장하는 단계.According to another aspect of the present invention, the present invention provides a method for preparing a pharmaceutical composition, comprising the following steps: (a) at least one of cell debris, waste matter, and giant particles by passing the culture medium of cord blood stem cells through a filter filtering; (b) sterilizing the filtered culture medium with a sterile filter; (c) separating exosomes from the sterilized culture solution through concentration and diafiltration using tangential flow filtration; (d) diluting the separated exosomes again to a starting volume by adding a stabilization buffer; and (e) sterilizing the diluted exosomes by passing them through a sterilization filter using a liquid exchange system and storing them in a sealed storage container.
본 발명의 일 구현예에 따르면, 상기 단계 (c)의 접선흐름여과방법은 분자량 한계치를 얻기 위하여 10,000 Da, 50,000 Da 또는 75,000 Da의 중공사 필터를 사용하는 것을 특징으로 한다.According to one embodiment of the present invention, the tangential flow filtration method of step (c) is characterized in that a hollow fiber filter of 10,000 Da, 50,000 Da or 75,000 Da is used to obtain a molecular weight limit.
본 발명의 일 구현예에 따르면, 상기 단계 (c)의 농축과 정용여과과정은 접선흐름여과를 단속 또는 연속적으로 진행하고, 농축은 시작 부피에 대하여 10배 내지 25배 농축하고, 정용여과과정은 농축된 부피에 대하여 적어도 10배의 부피를 갖는 버퍼로 여과하는 과정을 포함하는 것을 특징으로 한다.According to one embodiment of the present invention, in the concentration and diafiltration process of step (c), tangential flow filtration is performed intermittently or continuously, the concentration is concentrated 10 to 25 times with respect to the starting volume, and the diafiltration process is It is characterized in that it comprises the step of filtering with a buffer having a volume of at least 10 times with respect to the concentrated volume.
본 발명의 일 구현예에 따르면, 상기 단계 (d)의 희석단계는 농축 및 정용여과된 부피에 대하여 10배 내지 25배의 안정화 버퍼를 첨가하여 전체 부피가 시작 부피와 같도록 희석하는 과정을 포함하는 것을 특징으로 한다.According to one embodiment of the present invention, the dilution step of step (d) includes a process of diluting the total volume to be equal to the starting volume by adding a
본 발명은 제대혈간세포 유래 엑소좀을 유효성분으로 포함하는 창상 치료 또는 창상 치료 촉진용 약학적 조성물에 관한 것으로, 본 발명은 염증성 사이토카인의 생성 억제, 활성산소 증가의 감소 및 SOD(super oxide dismutase) 활성 감소의 효과적인 회복을 보였으며, 창상부위에서의 혈관손상으로 인한 면역세포 침윤현상의 회복, 상처 치유 및 세포 이동 효과에 의해 창상 치료가 촉진되는 특징을 가지므로서, 효과적인 창상 치료 목적으로 활용될 수 있다.The present invention relates to a pharmaceutical composition for treating wounds or promoting wound healing comprising exosomes derived from cord blood stem cells as an active ingredient, and the present invention is directed to inhibiting the production of inflammatory cytokines, reducing the increase in reactive oxygen species and super oxide dismutase (SOD) It showed effective recovery of activity reduction, recovery of immune cell infiltration caused by blood vessel damage in the wound site, wound healing and cell migration effect, promoting wound healing, so it can be used for effective wound treatment purposes. can
도 1은 본 발명의 엑소좀을 초저온전자현미경으로 분석한 결과이다.
도 2는 본 발명의 엑소좀을 인간 피부조직에 적용한 후 피부조직 생검을 수행하여 창상부위에서의 면역세포 침윤현상 억제 및 피부조직의 회복을 나타낸 것이다.
도 3은 본 발명의 엑소좀에 의한 (a) IL-6 및 (b) TNFα의 분비 변화량을 ELISA 키트로 확인한 결과를 나타낸 것이다.
도 4는 본 발명의 엑소좀에 의한 활성산소 감소 효과를 나타낸 것으로, 도 4a는 형광현미경으로 관찰한 이미지이며, 도 4b는 정량분석한 결과를 나타낸 것이다.
도 5는 본 발명의 엑소좀에 의한 항산화효소 SOD의 변화량을 ELISA 키트로 확인한 결과를 나타낸 것이다.
도 6은 본 발명의 엑소좀에 의해 상처 부위로 HaCaT 세포의 이동 변화를 나타낸 것으로, 도 6a는 현미경으로 관찰한 이미지이며, 도 6b는 scar 면적을 정량분석한 결과를 나타낸 것이다.
도 7은 본 발명의 엑소좀에 의해 피부세포 이동(migration) 효과를 나타낸 것으로, 도 7a는 현미경으로 관찰한 이미지이며, 도 7b는 이동한 피부세포를 Image J로 정량분석한 결과를 나타낸 것이다.1 is a result of analyzing the exosome of the present invention with a cryo-electron microscope.
Figure 2 shows the inhibition of immune cell infiltration and recovery of skin tissue in the wound site by performing a skin tissue biopsy after applying the exosome of the present invention to human skin tissue.
Figure 3 shows the results of confirming the amount of secretion of (a) IL-6 and (b) TNFα by the exosomes of the present invention using an ELISA kit.
Figure 4 shows the active oxygen reduction effect by the exosomes of the present invention, Figure 4a is an image observed with a fluorescence microscope, Figure 4b shows the results of quantitative analysis.
Figure 5 shows the results of confirming the amount of change in antioxidant enzyme SOD by the exosomes of the present invention with an ELISA kit.
Figure 6 shows the change in migration of HaCaT cells to the wound site by the exosomes of the present invention, Figure 6a is an image observed under a microscope, and Figure 6b shows the result of quantitative analysis of the scar area.
Figure 7 shows the skin cell migration (migration) effect by the exosomes of the present invention, Figure 7a is an image observed under a microscope, Figure 7b shows the result of quantitative analysis of the migrated skin cells with Image J.
본 명세서에서 설명된 구체적인 실시예는 본 발명의 바람직한 구현예 또는 예시를 대표하는 의미이며, 이에 의해 본 발명의 범위가 한정되지는 않는다. 본 발명의 변형과 다른 용도가 본 명세서 특허청구범위에 기재된 발명의 범위로부터 벗어나지 않는다는 것은 당업자에게 명백하다.Specific examples described in this specification are meant to represent preferred embodiments or examples of the present invention, and the scope of the present invention is not limited thereby. It will be apparent to those skilled in the art that variations and other uses of the present invention do not depart from the scope of the invention described in the claims.
[실시예 1] 제대혈간세포 수득 및 배양[Example 1] Obtaining and culturing cord blood stem cells
1) 제대혈간세포 수득1) Obtaining cord blood stem cells
기증자로부터 얻은 제대혈을 12시간 내에 처리한다. 구체적인 처리과정은 아래와 같다. 제대혈을 50ml 용량의 원추형 튜브에 옮긴 후, 원추형 튜브를 원심분리기를 이용하여 실온조건에서 2000rpm의 속도로 30분 동안 원심분리하였다. 원심분리 후 상층액(혈장)을 제거하고 흰색으로 보이는 완충층(buffy coat layer)을 수거한다. 수거한 완충층을 피콜(Ficoll)이 담겨있는 50ml 용량의 원추형 튜브에 피콜과 완충층의 비율이 2 : 1이 되도록 완충층을 천천히 주입한다. 피콜과 완충층이 혼합된 원추형 튜브를 원심분리기를 이용하여 실온조건에서 2000rpm의 회전속도로 30분동안 원심분리하였다. 원심분리 후 상층액(혈장)을 제거하고 흰색으로 보이는 단핵세포층(mononuclear cell layer)을 수거하였다. 수거한 단핵세포를 1배 인산완충생리식염수(1xPBS, 칼슘과 마그네슘이 미포함)에 충분히 혼합하고 10μl의 샘플을 채취하여 자동 형광세포 카운터(LUNA STEM장비)로 셀 카운팅을 진행하였다.Cord blood obtained from a donor should be processed within 12 hours. The specific processing process is as follows. After transferring the cord blood to a conical tube with a capacity of 50 ml, the conical tube was centrifuged for 30 minutes at a speed of 2000 rpm at room temperature using a centrifuge. After centrifugation, the supernatant (plasma) is removed and the white buffy coat layer is collected. The collected buffer layer is slowly injected into a 50 ml conical tube containing Ficoll so that the ratio of Ficoll to buffer layer is 2:1. The conical tube in which Ficoll and the buffer layer were mixed was centrifuged for 30 minutes at a rotational speed of 2000 rpm at room temperature using a centrifuge. After centrifugation, the supernatant (plasma) was removed, and the mononuclear cell layer, which appeared white, was collected. The collected mononuclear cells were sufficiently mixed with 1x phosphate buffered saline (1xPBS, without calcium and magnesium), and a 10 μl sample was taken and cell counting was performed with an automatic fluorescent cell counter (LUNA STEM equipment).
한랭쇼크 방법에 의한 제대혈간세포를 수득하기 위해서, 단핵세포를 포함하는 혼합액은 원심분리기를 이용하여 실온조건에서 1500rpm의 회전속도에서 5분 동안 원심분리한 후, 상층액을 제거하고 10% 녹아웃 혈청 대체물(Knockout serum replacement)과 2mM의 글루타민(Glutamine)이 포함된 DMEM/F-12배지로 단핵세포를 재부유시켜 10% 디메틸술폭시드(DMSO : Dimethyl sulfoxide)를 추가한 후 충분히 혼합하여 저온 유리병(cryo vial)에 분주하여 냉동 컨테이너에 넣고 -80℃ 초저온 냉장고(deep freezer)에서 24시간 냉동보관한 후, 37℃ 항온수조에서 5분 동안 급속 해동하여 제대혈간세포를 수득하였다.In order to obtain umbilical cord blood stem cells by the cold shock method, the mixed solution containing mononuclear cells was centrifuged for 5 minutes at a rotational speed of 1500 rpm at room temperature using a centrifuge, and then the supernatant was removed and 10% knockout serum substitute was added. (Knockout serum replacement) and mononuclear cells were resuspended in DMEM/F-12 medium containing 2mM glutamine, 10% dimethyl sulfoxide (DMSO) was added, mixed sufficiently, and stored in a low-temperature glass bottle ( cryo vial), placed in a freezing container, frozen in a -80°C deep freezer for 24 hours, and rapidly thawed in a 37°C constant temperature water bath for 5 minutes to obtain cord blood stem cells.
2) 제대혈간세포 배양2) Umbilical Cord Blood Stem Cell Culture
수득한 제대혈간세포를 차가운 1배 인산완충생리식염수에 충분히 혼합하고 40μm 셀 스트레이너(cell strainer)를 통과하여 뭉쳐져 있을 수 있는 세포를 제거한 후 10μl의 샘플을 채취하여 자동 형광세포 카운터로 셀 카운팅을 진행하였고, 나머지 샘플은 원심분리기로 상온에서 1200rpm의 회전속도에서 10분동안 원심분리한 후, 상층액을 제거하고 20% 우태아혈청(FBS : fetal bovine serum)을 함유한 배지(Advanced DMEM 사용)로 재부유시켜 세포배양접시에 접종한 후 5% CO2, 37℃ 조건 하에서 배양하였다. 75% 이상의 제대혈간세포가 부착된 후, 1배 인산완충생리식염수로 충분히 세척 후, 10% 녹아웃 혈청 대체물(Knockout serum replacement), 2mM L-글루타민의 디펩타이드 형태(L-alanyl-L-glutamine dipeptide)를 함유한 DMEM/F-12 배지로 교체하여 4주 내지 5주간 배양하고 그 배양액을 회수하였다. The obtained umbilical cord blood stem cells were sufficiently mixed with cold 1x phosphate buffered saline, passed through a 40 μm cell strainer to remove cells that could be clumped, and then a 10 μl sample was taken and cell counting was performed with an automatic fluorescent cell counter. , The remaining samples were centrifuged for 10 minutes at a rotational speed of 1200 rpm at room temperature in a centrifuge, and then the supernatant was removed and reconstituted with a medium (Advanced DMEM) containing 20% fetal bovine serum (FBS). After being suspended and inoculated into a cell culture dish, the cells were cultured under 5% CO 2 , 37°C conditions. After more than 75% of umbilical cord blood stem cells adhere, wash thoroughly with 1x phosphate buffered saline, 10% knockout serum replacement, 2 mM L-glutamine dipeptide (L-alanyl-L-glutamine dipeptide) was replaced with DMEM/F-12 medium containing , cultured for 4 to 5 weeks, and the culture medium was recovered.
본 발명의 방법으로 부착한 제대혈간세포의 세포표면 표지자 CD24, CD31, CD45RA, CD105, CD117, CD146, SSEA-1 및 TRA-1-60를 Thermo Fisher Scientific사의 유세포 분석기로 분석하였고 그 결과를 표 1에 도시하였다.The cell surface markers CD24, CD31, CD45RA, CD105, CD117, CD146, SSEA-1 and TRA-1-60 of cord blood stem cells attached by the method of the present invention were analyzed using a flow cytometer from Thermo Fisher Scientific, and the results are shown in Table 1. shown
[실시예 2] 접선흐름여과를 이용한 엑소좀 분리[Example 2] Separation of exosomes using tangential flow filtration
상기 제대혈간세포 배양액에 함유된 엑소좀은 농도가 높고, 입자크기 분포가 균일하기에 접선흐름여과 시스템으로 엑소좀을 분리하기 용이하고 화학물질 또는 소분자 물질 등 첨가물을 추가하지 않았다. 우선 배양액을 20ml/min의 속도로 펌핑하여 필터(Sartopore 2 XLG SartoScale 25(0.8μm, 0.2μm) 사용)로 여과하여 세포 잔해물, 노폐물 및 거대입자 등 불순물을 제거하고 멸균까지 진행하였다. 여과멸균된 배양액은 즉시 또는 -20℃ 냉동고에 동결보관하였다가 해동시킨 후, 접선흐름여과(TFF: Tangential Flow Filtration)방법을 이용하여 엑소좀을 분리하였다. Since the exosomes contained in the cord blood stem cell culture medium have a high concentration and a uniform particle size distribution, it is easy to separate the exosomes using a tangential flow filtration system, and additives such as chemicals or small molecules are not added. First, the culture medium was pumped at a rate of 20 ml/min and filtered with a filter (
상기 여과멸균된 배양액에서 엑소좀을 분리하기 위하여 접선흐름여과방법으로 농축 및 정용여과(diafiltration)를 진행하였다. 접선흐름여과방법을 위한 필터로는 중공사 필터(Repligen에서 구매)를 사용하였다. 중공사 필터는 엑소좀의 성질과 여과 목적에 따라 다양한 재질과 분자량 한계치(Molecular weight cutoff)을 선택할 수 있다. 선택된 분자량 한계치에 의해 선별적으로 엑소좀을 분리하였고 선택된 분자량 한계치보다 작은 입자나 단백질, 지질, 핵산, 저분자 화합물 등은 모두 제거하였다. 엑소좀을 분리하기 위한 중공 섬유로는 mPES(Modified Polyethersulfone), PS(Polysulfone), PES(Polyethersulfone) 또는 ME(Mixed Cellulose Ester)재질을 사용하였고, 분자량 한계치는 10,000 Da, 50,000 Da 또는 75,000 Da를 사용하였다. 접선흐름여과방법을 이용한 농축 및 정용여과(diafiltration)과정은 단속 또는 연속적으로 수행하였고 농축은 시작 부피에 대하여 10배 내지 25배 농축하고, 정용여과는 농축한 부피에 대하여 적어도 10배, 바람직하게는 12배 내지 15배 이상의 부피를 갖는 완충용액을 이용하여 수행하였다. 완충용액은 1배 인산완충생리식염수(1xPBS, 칼슘과 마그네슘이 미포함, Corning에서 구매)를 사용하였고 어떠한 화학물질 또는 저분자 물질도 첨가하지 않았다. 접선흐름여과방법으로 농축과 정용여과를 거친 엑소좀은 안정화 버퍼를 10배 내지 25배 추가하여 엑소좀의 부피가 시작 부피와 같게 되도록 희석하였다.In order to isolate the exosomes from the filter-sterilized culture medium, concentration and diafiltration were performed using a tangential flow filtration method. A hollow fiber filter (purchased from Repligen) was used as a filter for the tangential flow filtration method. The hollow fiber filter can be selected from various materials and molecular weight cutoffs depending on the nature of exosomes and the purpose of filtration. Exosomes were selectively separated according to the selected molecular weight threshold, and all particles, proteins, lipids, nucleic acids, and low-molecular-weight compounds smaller than the selected molecular weight threshold were removed. mPES (Modified Polyethersulfone), PS (Polysulfone), PES (Polyethersulfone) or ME (Mixed Cellulose Ester) materials were used as hollow fibers for separating exosomes, and molecular weight limits of 10,000 Da, 50,000 Da, or 75,000 Da were used. did The concentration and diafiltration process using the tangential flow filtration method was performed intermittently or continuously, and the concentration was concentrated 10 to 25 times the starting volume, and the diafiltration was at least 10 times the concentrated volume, preferably. It was performed using a buffer solution having a volume of 12 to 15 times or more. As the buffer solution, 1x phosphate buffered saline (1xPBS, without calcium and magnesium, purchased from Corning) was used, and no chemicals or low-molecular substances were added. After concentration and diafiltration by the tangential flow filtration method, the exosomes were diluted by adding a
본 발명의 방법으로 분리한 엑소좀을 최종적으로 0.2 μm Sartopore 2 XLG Caps(Sartorius에서 구매)로 여과 멸균하고 바로 이어지는 일회용 조립 밀폐용기의 포트(Thermo Fisher Scientific에서 구매)를 통해 완전한 무균상태가 보장된 밀폐용기에 저장하여 수송하였다.The exosomes isolated by the method of the present invention are finally filtered and sterilized with 0.2
상기 기술한 방법으로 배양액에서 30 내지 200 nm 균일한 입자크기의 엑소좀을 분리하였다. Exosomes having a uniform particle size of 30 to 200 nm were isolated from the culture medium by the method described above.
[실시예 3] 분리된 제대혈간세포 유래 엑소좀의 특성 분석[Example 3] Characterization of isolated exosomes derived from cord blood stem cells
본 발명의 제대혈간세포 유래 엑소좀의 모양을 확인하기 위하여 접선흐름여과장치로 농축한 엑소좀을 total exosome isolation kit(Thermo Fisher Scientific에서 구매)으로 침전한 후 1배 인산완충생리식염수(1xPBS)로 재현탁하여 초저온전자현미경 분석(서울대학교 농생명과학공동기기원)을 진행하였고 그 결과는 도 1과 같다. In order to confirm the shape of the exosomes derived from umbilical cord blood stem cells of the present invention, the exosomes concentrated with a tangential flow filtration device were precipitated with a total exosome isolation kit (purchased from Thermo Fisher Scientific), and then reproduced with 1x phosphate buffered saline (1xPBS). Cryogenic electron microscopy analysis (Seoul National University Agricultural Life Science Joint Institute) was conducted, and the results are shown in FIG. 1.
또한, 상기 분리된 제대혈간세포 유래 엑소좀을 100mM TEAB와 1% SDS가 함유된 버퍼로 재현탁하여 충분히 반응시킨 후 원심분리하여 엑소좀 단백질을 획득하였다. 다음, 엑소좀 단백질을 TMT Mass Tagging Kits and Reagents(Thermo Fisher Scientific에서 구매)로 라벨링하고 High pH Reversed-Phase Peptide Fractionation Kit(Thermo Fisher Scientific에서 구매)로 정제하여 Orbitrap LC-MS(KBSI 위탁 분석)로 분석하였다. 그 결과 본 발명의 엑소좀은 표면마커에 대해, CD9, CD63, CD81 외에 CD44, CD53 또는 CD98를 포함하는 것으로 확인되었다.In addition, the isolated exosomes derived from cord blood stem cells were resuspended in a buffer containing 100 mM TEAB and 1% SDS, sufficiently reacted, and then centrifuged to obtain exosome proteins. Next, exosomal proteins were labeled with TMT Mass Tagging Kits and Reagents (purchased from Thermo Fisher Scientific), purified with a High pH Reversed-Phase Peptide Fractionation Kit (purchased from Thermo Fisher Scientific), and analyzed by Orbitrap LC-MS (KBSI Commissioned Analysis). analyzed. As a result, it was confirmed that the exosomes of the present invention contain CD44, CD53, or CD98 in addition to CD9, CD63, and CD81 for surface markers.
[실시예 4] HaCaT 세포의 배양[Example 4] Culture of HaCaT cells
인간 피부세포주인 HaCaT 세포를 10% 우태아혈청(Fetal bovine serum, ThermoFisher Scientific에서 구매), 2 mM L-글루타민(ThermoFisher Scientific에서 구매) 및 1% 항생제-항진균제(Antibiotics-antimyotics, ThermoFisher Scientific에서 구매)가 함유된 DMEM(ThermoFisher Scientific에서 구매) 배지에 5% CO2, 37℃ 조건에서 계대배양하였다.HaCaT cells, a human skin cell line, were cultured in 10% fetal bovine serum (purchased from ThermoFisher Scientific), 2 mM L-glutamine (purchased from ThermoFisher Scientific), and 1% antibiotics-antimyotics (purchased from ThermoFisher Scientific). DMEM (purchased from ThermoFisher Scientific) medium containing 5% CO 2 , and subcultured at 37°C.
[실시예 5] 인간 피부조직 생검을 통한 엑소좀 효과 분석[Example 5] Analysis of exosome effect through human skin tissue biopsy
본 발명의 일 실시예의 분리방법에 따라 수득된 엑소좀을 함유하는 조성물을 정상인의 피부에 창상을 유발하여 1시간 도포한 후 정상피부, 창상피부 및 창상피부에 상기 엑소좀을 처리한 피부조직에 대한 생검을 실시하였다. A composition containing the exosomes obtained according to the separation method of an embodiment of the present invention is applied to the skin of a normal person for 1 hour by inducing a wound, and then applied to normal skin, wound skin, and skin tissue treated with the exosomes. A biopsy was performed.
그 결과, 창상부위에서 혈관손상으로 인한 면역세포 침윤현상이 회복되었고 침윤된 면역세포가 제거되었으며 혈관이 복구되었다(도 2).As a result, immune cell infiltration caused by blood vessel damage was restored at the wound site, infiltrated immune cells were removed, and blood vessels were restored (FIG. 2).
따라서, 본 발명의 일 실시예의 분리방법에 따라 수득된 엑소좀을 유효성분으로 함유하는 창상 치료 조성물은 상기와 같은 임상시험을 통해 창상피부 조직에서 발생하는 혈관주위 면역세포 침윤현상에 대해 치료효과를 나타내는 것으로 확인할 수 있었다.Therefore, the wound treatment composition containing the exosomes obtained according to the separation method of an embodiment of the present invention as an active ingredient has a therapeutic effect on the infiltration of perivascular immune cells occurring in wound skin tissue through the clinical trials as described above. It could be confirmed by showing
[실시예 6] 본 발명의 엑소좀의 항염증 효과 시험[Example 6] Anti-inflammatory effect test of the exosome of the present invention
1) 염증성 사이토카인(IL-6 및 TNFα)의 분비 변화량 분석1) Analysis of changes in secretion of inflammatory cytokines (IL-6 and TNFα)
HaCaT 세포를 10% 우태아혈청, 2mM L-글루타민을 포함한 DMEM 배지에 현탁시키고 이를 배양접시에 30~40%의 밀집도를 갖도록 분주하였다. 다음날 LPS가 포함된 새로운 배지로 염증반응을 유발한 후 본 발명의 엑소좀(Timebio exosome)과 지방 유래 중간엽줄기세포 엑소좀(MSC exosome)을 각각 24시간동안 처리하여 배양하였다. 배양이 끝난 후 세포파쇄물과 배양상층액을 취하여 그 속에 존재하는 IL-6 및 TNFα(ThermoFisher Scientific에서 구매)의 분비 변화량을 ELISA kit로 측정하였다.HaCaT cells were suspended in DMEM medium containing 10% fetal bovine serum and 2mM L-glutamine, and then seeded in a culture dish to have a confluency of 30 to 40%. The next day, after inducing an inflammatory response with a new medium containing LPS, the exosome (Timebio exosome) and the adipose-derived mesenchymal stem cell exosome (MSC exosome) of the present invention were treated and cultured for 24 hours, respectively. After the culture was completed, the cell lysate and the culture supernatant were taken, and the changes in secretion of IL-6 and TNFα (purchased from ThermoFisher Scientific) present therein were measured using an ELISA kit.
그 결과, 본 발명의 엑소좀은 HaCaT 세포에서 LPS에 의해 유도되는 IL-6(도 3a) 및 TNFα(도 3b) 사이토카인의 생성을 효과적으로 감소시키는 것을 확인하였고, 또한 그 효과는 지방 유래 중간엽줄기세포 엑소좀(MSC exosome)에 비해서도 우수함을 확인하였다(도 3).As a result, it was confirmed that the exosomes of the present invention effectively reduce the production of cytokines IL-6 (Fig. 3a) and TNFα (Fig. 3b) induced by LPS in HaCaT cells, and the effect was also found to be effective in adipose-derived mesenchymal It was confirmed that it was superior to the stem cell exosome (MSC exosome) (FIG. 3).
2) 활성산소 분석2) Active oxygen analysis
HaCaT 세포를 10% 우태아혈청, 2mM L-글루타민을 포함한 DMEM 배지에 현탁시키고 이를 배양접시에 30~40%의 밀집도를 갖도록 분주하였다. 다음날 LPS가 포함된 새로운 배지로 염증반응을 유발한 후 본 발명의 엑소좀(Timebio exosome), 지방 유래 중간엽줄기세포 엑소좀(MSC exosome) 및 NAC (N-acetyl cysteine, 양성 대조군)을 각각 24시간동안 처리하여 배양한 후 활성산소의 변화량을 CellROX Green Reagent (ThermoFisher Scientific에서 구매)로 염색하여 형광현미경으로 관찰하였으며(도 4a), 이후 활성산소에 대한 정량분석을 시행하였다(도 4b).HaCaT cells were suspended in DMEM medium containing 10% fetal bovine serum and 2mM L-glutamine, and then seeded in a culture dish to have a confluency of 30 to 40%. The next day, after inducing an inflammatory response with a new medium containing LPS, the exosome of the present invention (Timebio exosome), adipose-derived mesenchymal stem cell exosome (MSC exosome), and NAC (N-acetyl cysteine, positive control) were treated at 24 After culturing for a period of time, the amount of change in active oxygen was stained with CellROX Green Reagent (purchased from ThermoFisher Scientific) and observed under a fluorescence microscope (FIG. 4a), followed by quantitative analysis of active oxygen (FIG. 4b).
그 결과, 본 발명의 엑소좀은 HaCaT 세포에서 LPS에 의해 유도되는 활성산소의 증가를 효과적으로 감소시키는 것을 확인하였고, 또한 그 효과는 지방 유래 중간엽줄기세포 엑소좀(MSC exosome)에 비해서도 우수함을 확인하였다(도 4).As a result, it was confirmed that the exosome of the present invention effectively reduces the increase in reactive oxygen species induced by LPS in HaCaT cells, and that the effect is superior to adipose-derived mesenchymal stem cell exosome (MSC exosome) (FIG. 4).
3) SOD(Super Oxide Dismutase) 활성 분석3) SOD (Super Oxide Dismutase) activity analysis
HaCaT 세포를 10% 우태아혈청, 2mM L-글루타민을 포함한 DMEM 배지에 현탁시키고 이를 배양접시에 30~40%의 밀집도를 갖도록 분주하였다. 다음날 LPS가 포함된 새로운 배지로 염증반응을 유발한 후 본 발명의 엑소좀(Timebio exosome), 지방 유래 중간엽줄기세포 엑소좀(MSC exosome) 및 NAC (N-acetyl cysteine, 양성 대조군)을 각각 24시간동안 처리하여 배양한 후 SOD활성(ThermoFisher Scientific에서 구매)의 변화량을 ELISA kit로 측정하였다. HaCaT cells were suspended in DMEM medium containing 10% fetal bovine serum and 2mM L-glutamine, and then seeded in a culture dish to have a confluency of 30 to 40%. The next day, after inducing an inflammatory response with a new medium containing LPS, the exosome of the present invention (Timebio exosome), adipose-derived mesenchymal stem cell exosome (MSC exosome), and NAC (N-acetyl cysteine, positive control) were treated at 24 After culturing for a period of time, the amount of change in SOD activity (purchased from ThermoFisher Scientific) was measured using an ELISA kit.
그 결과, 본 발명의 엑소좀은 HaCaT 세포에서 LPS에 의해 유도되는 SOD 활성 감소를 효과적으로 회복시킨다는 것을 확인하였고, 또한 그 효과는 지방 유래 중간엽줄기세포 엑소좀(MSC exosome)에 비해서도 우수함을 확인하였다(도 5).As a result, it was confirmed that the exosome of the present invention effectively restores the decrease in SOD activity induced by LPS in HaCaT cells, and that the effect is superior to adipose-derived mesenchymal stem cell exosome (MSC exosome). (FIG. 5).
상기 결과들은 본 발명의 엑소좀이 창상부위에서의 염증 관련 반응을 조절하여 창상 치료 조성물의 유효성분으로 활용될 수 있음을 보여주며, 지방 유래 중간엽줄기세포 엑소좀(MSC exosome)보다 염증 완화 효능이 뛰어나므로, 창상 치료 조성물의 유효성분으로 사용될 경우, 종래 중간엽줄기세포 유래 엑소좀보다 더욱 우수한 창상 치료 효과를 기대할 수 있다. The above results show that the exosome of the present invention can be used as an active ingredient in a wound treatment composition by regulating the inflammation-related response at the wound site, and has a more inflammatory relieving effect than adipose-derived mesenchymal stem cell exosome (MSC exosome) Since it is excellent, when used as an active ingredient of a wound treatment composition, a more excellent wound treatment effect than conventional mesenchymal stem cell-derived exosomes can be expected.
[실시예 7] 본 발명의 엑소좀의 상처치유 효과 시험[Example 7] Wound healing effect test of the exosome of the present invention
HaCaT 세포를 10% 우태아혈청, 2mM L-글루타민을 포함한 DMEM 배지에 현탁시키고 이를 Scar TM Block (SPL에서 구매) 배양접시에 30~40%의 밀집도를 갖도록 분주하였다. 다음날 scar block을 제거하고(상처 부위 형성) 본 발명의 엑소좀과 지방 유래 중간엽줄기세포 엑소좀(MSC exosome)좀을 각각 처리하여 18시간 또는 24시간 후에 세포 이동 정도를 현미경으로 관찰하였고(도 6a), scar 면적을 Image J로 정량분석을 시행하였다(도 6b). HaCaT cells were suspended in DMEM medium containing 10% fetal calf serum and 2mM L-glutamine, and seeded onto Scar TM Block (purchased from SPL) culture dishes to have a confluency of 30-40%. The next day, the scar block was removed (formation of scar), and the exosome of the present invention and the adipose-derived mesenchymal stem cell exosome (MSC exosome) were treated, respectively, and the degree of cell migration was observed under a microscope after 18 or 24 hours (Fig. 6a), the scar area was quantitatively analyzed with Image J (FIG. 6b).
그 결과, 본 발명의 엑소좀을 24시간 처리한 경우 HaCaT 세포에서의 상처 부위로 많은 세포가 이동하여 상처 부위가 완전히 세포로 메워짐을 확인하였고, 또한 그 효과는 지방 유래 중간엽줄기세포 엑소좀(MSC exosome)에 비해서도 우수함을 확인하였다(도 6). As a result, when the exosome of the present invention was treated for 24 hours, it was confirmed that many cells migrated to the wound site in HaCaT cells and the wound site was completely filled with cells. MSC exosome) was also confirmed to be superior (FIG. 6).
[실시예 8] 본 발명의 엑소좀의 세포이동 효과 시험[Example 8] Test of cell migration effect of exosomes of the present invention
HaCaT 세포를 10% 우태아혈청, 2mM L-글루타민을 포함한 DMEM 배지에 현탁시키고 이를 트랜스웰 플레이트(Transwell plate, Corning에서 구매)에 30~40%의 밀집도를 갖도록 분주하였다. 다음날 트랜스웰 플레이트에 본 발명의 엑소좀과 지방 유래 중간엽줄기세포 엑소좀(MSC exosome)을 각각 24시간동안 처리하여 배양하였다. 배양이 끝난 후, 트랜스웰을 취하여 크리스탈 바이올렛 용액(Crystal violet solution, Sigma에서 구매)으로 염색한 후 트랜스웰로 이동하여 염색된 세포를 실체현미경으로 관찰하였고(도 7a), 염색된 세포(이동한 세포)를 Image J로 정량분석을 시행하였다(도 7b).HaCaT cells were suspended in DMEM medium containing 10% fetal bovine serum and 2 mM L-glutamine, and then seeded to a confluency of 30-40% on a Transwell plate (Transwell plate, purchased from Corning). The next day, the exosome of the present invention and the adipose-derived mesenchymal stem cell exosome (MSC exosome) were respectively treated and cultured in a transwell plate for 24 hours. After incubation, the transwell was taken, stained with crystal violet solution (purchased from Sigma), moved to the transwell, and the stained cells were observed under a stereomicroscope (Fig. 7a), and the stained cells (transferred cells) was subjected to quantitative analysis with Image J (FIG. 7b).
그 결과, 본 발명의 엑소좀을 24시간 처리한 경우 트랜스웰로 이동한 HaCaT 세포 수가 대조군(control)에 비해 2.5배 이상 증가되었음을 확인하였고, 또한 그 효과는 지방 유래 중간엽줄기세포 엑소좀(MSC exosome)에 비해서도 우수함을 확인하였다(도 7). 상기 본 발명의 엑소좀이 가지는 상처치유 효과와 세포이동 효과를 통해, 본 발명의 엑소좀이 창상부위에서의 염증반응을 조절할 뿐만 아니라, 피부세포의 상처치유와 세포이동을 촉진할 수 있음을 확인할 수 있었고, 또한 이러한 효과는 지방 유래 중간엽줄기세포 엑소좀(MSC exosome)에 비해서 현저히 우수함을 확인하였다.As a result, when the exosome of the present invention was treated for 24 hours, it was confirmed that the number of HaCaT cells migrating to the transwell increased by more than 2.5 times compared to the control group, and the effect was also confirmed by adipose-derived mesenchymal stem cell exosomes (MSC exosome) was also confirmed to be superior (FIG. 7). Through the wound healing effect and cell migration effect of the exosome of the present invention, it is confirmed that the exosome of the present invention can promote wound healing and cell migration of skin cells as well as regulate the inflammatory response in the wound site. It was also confirmed that this effect was significantly superior to adipose-derived mesenchymal stem cell exosome (MSC exosome).
이상으로 본 발명의 특정한 부분을 상세히 기술하였는 바, 당업계의 통상의 지식을 가진 자에게 있어서 이러한 구체적인 기술은 단지 바람직한 구현 예일뿐이며, 이에 본 발명의 범위가 제한되는 것이 아닌 점은 명백하다. 따라서, 본 발명의 실질적인 범위는 첨부된 청구항과 그의 등가물에 의하여 정의된다고 할 것이다.Since the specific parts of the present invention have been described in detail above, it is clear that these specific techniques are only preferred embodiments for those skilled in the art, and the scope of the present invention is not limited thereto. Accordingly, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.
Claims (14)
A pharmaceutical composition for wound healing or promoting wound healing comprising exosomes derived from cord blood progenitor cells as an active ingredient.
상기 제대혈간세포는 표면마커 중에서 CD24, CD117 및 SSEA-1에 대하여 음성의 면역학적 특성을 나타내고, CD31, CD45RA, CD105, CD146 또는 TRA-1-60에 대하여 양성의 면역학적 특성을 나타내는 것을 특징으로 하는 창상 치료 또는 창상 치료 촉진용 약학적 조성물.
According to claim 1,
The cord blood stem cells exhibit negative immunological properties for CD24, CD117 and SSEA-1 among surface markers, and positive immunological properties for CD31, CD45RA, CD105, CD146 or TRA-1-60 Characterized in that A pharmaceutical composition for wound healing or promoting wound healing.
상기 엑소좀은 표면마커 중에서 CD9, CD63, CD81 외에 CD44, CD53 또는 CD98을 더 포함하는 것을 특징으로 하는 창상 치료 또는 창상 치료 촉진용 약학적 조성물.
According to claim 1,
The exosome is a pharmaceutical composition for wound healing or promoting wound healing, characterized in that it further comprises CD44, CD53 or CD98 in addition to CD9, CD63, CD81 among surface markers.
상기 조성물은 정제수, 폴리올, 히알루론산 및 방부제으로 이루어진 그룹에서 선택된 적어도 하나의 보조성분을 더 포함하는 것을 특징으로 하는 창상 치료 또는 창상 치료 촉진용 약학적 조성물.
According to claim 1,
The composition further comprises at least one auxiliary ingredient selected from the group consisting of purified water, polyol, hyaluronic acid and an antiseptic, or a pharmaceutical composition for wound healing or promoting wound healing.
A pharmaceutical formulation for wound healing or promoting wound healing comprising the pharmaceutical composition of any one of claims 1 to 4.
상기 제제는 주사제형, 주입제형, 분무제형, 액상제형 또는 패취제형인 것을 특징으로 하는 창상 치료 또는 창상 치료 촉진용 약학 제제.
According to claim 5,
The pharmaceutical formulation for wound healing or promoting wound healing, characterized in that the formulation is in the form of an injection, infusion, spray, liquid or patch formulation.
A quasi-drug preparation for wound improvement comprising the pharmaceutical composition of any one of claims 1 to 4.
상기 의약외품 제제는 액제, 연고제, 크림제, 스프레이제, 패취제, 겔제 및 에어로졸제로 이루어지는 군으로부터 선택되는 피부 외용제 형태인 것을 특징으로 하는 의약외품 제제.
According to claim 7,
The quasi-drug formulation is a quasi-drug formulation, characterized in that in the form of an external skin preparation selected from the group consisting of solutions, ointments, creams, sprays, patches, gels and aerosols.
A medical device for wound healing or promoting wound healing comprising the pharmaceutical composition of any one of claims 1 to 4.
상기 의료기기는 창상 피복재 또는 지혈용 기재인 것을 특징으로 하는 의료기기.
According to claim 9,
The medical device is characterized in that the wound covering material or base material for hemostasis.
(a) 제대혈간세포 배양액을 필터에 통과시켜 세포 잔해물, 노폐물, 거대 입자 중 적어도 어느 하나를 여과하는 단계;
(b) 상기 여과된 배양액을 멸균 필터로 멸균하는 단계;
(c) 상기 멸균된 배양액을 접선흐름여과방법을 이용하여 농축과 정용여과과정을 통해 엑소좀을 분리하는 단계;
(d) 상기 분리된 엑소좀에 안정화 버퍼를 추가하여 다시 시작 부피가 되도록 희석하는 단계; 및
(e) 상기 희석된 엑소좀을 액체 교환 시스템으로 멸균 필터에 통과시켜 멸균한 후 밀봉된 저장용기에 저장하는 단계.
A method for preparing the pharmaceutical composition according to any one of claims 1 to 4, comprising the following steps:
(a) filtering at least one of cell debris, waste products, and giant particles by passing the culture medium of cord blood stem cells through a filter;
(b) sterilizing the filtered culture medium with a sterile filter;
(c) separating exosomes from the sterilized culture solution through concentration and diafiltration using tangential flow filtration;
(d) diluting the separated exosomes to a starting volume by adding a stabilization buffer; and
(e) sterilizing the diluted exosomes by passing them through a sterilizing filter using a liquid exchange system and storing them in a sealed storage container.
상기 단계 (c)의 접선흐름여과방법은 분자량 한계치를 얻기 위하여 10,000 Da, 50,000 Da 또는 75,000 Da의 중공사 필터를 사용하는 것을 특징으로 하는 제조방법.
According to claim 11,
In the tangential flow filtration method of step (c), a hollow fiber filter of 10,000 Da, 50,000 Da or 75,000 Da is used to obtain a molecular weight limit.
상기 단계 (c)의 농축과 정용여과과정은 접선흐름여과를 단속 또는 연속적으로 진행하고, 농축은 시작 부피에 대하여 10배 내지 25배 농축하고, 정용여과과정은 농축된 부피에 대하여 적어도 10배의 부피를 갖는 버퍼로 여과하는 과정을 포함하는 것을 특징으로 하는 제조방법.
According to claim 11,
In step (c), the concentration and diafiltration process is carried out intermittently or continuously by tangential flow filtration, the concentration is 10 to 25 times the starting volume, and the diafiltration process is at least 10 times the concentrated volume. A manufacturing method comprising the step of filtering with a buffer having a volume.
상기 단계 (d)의 희석단계는 농축 및 정용여과된 부피에 대하여 10배 내지 25배의 안정화 버퍼를 첨가하여 전체 부피가 시작 부피와 같도록 희석하는 과정을 포함하는 것을 특징으로 하는 제조방법.According to claim 11,
The dilution step of step (d) comprises a step of diluting the total volume to be equal to the starting volume by adding a stabilization buffer 10 to 25 times the concentrated and diafiltered volume.
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| KR20190012120A (en) | 2017-07-26 | 2019-02-08 | (주)유레 | Wound dressing comprising hyaluronic acid-calcium and polylysine, and preparation method thereof |
| KR20210063076A (en) | 2019-11-22 | 2021-06-01 | (주)메디팁 | wound dressing material for hemostasis and wound treatment, and method for preparing same |
| KR102271980B1 (en) | 2020-11-30 | 2021-07-02 | 주식회사 피엘마이크로메드 | Collagen-arginate wound dressing and method of preparing the same |
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| KR20190012120A (en) | 2017-07-26 | 2019-02-08 | (주)유레 | Wound dressing comprising hyaluronic acid-calcium and polylysine, and preparation method thereof |
| KR20210063076A (en) | 2019-11-22 | 2021-06-01 | (주)메디팁 | wound dressing material for hemostasis and wound treatment, and method for preparing same |
| KR102271980B1 (en) | 2020-11-30 | 2021-07-02 | 주식회사 피엘마이크로메드 | Collagen-arginate wound dressing and method of preparing the same |
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