JP2013523774A - Cell therapy to improve wound healing and related compositions and methods - Google Patents
Cell therapy to improve wound healing and related compositions and methods Download PDFInfo
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- JP2013523774A JP2013523774A JP2013502888A JP2013502888A JP2013523774A JP 2013523774 A JP2013523774 A JP 2013523774A JP 2013502888 A JP2013502888 A JP 2013502888A JP 2013502888 A JP2013502888 A JP 2013502888A JP 2013523774 A JP2013523774 A JP 2013523774A
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Abstract
創傷治癒を改善するための治療用組成物並びに関連する形成の方法及び使用を提供する。前記治療用組成物は生物分解性マトリックス、ゲル、又は皮下注射に適した溶液のいずれか一つに接種された適用量のＭＳＣを含む。前記適用量のＭＳＣは、投与毎に約１×１０6ＭＳＣ〜約１×１０10の範囲であってよい。投与体制は毎日、毎週、又は毎月であってよい。適用量及び治療間隔は、治療への患者の反応に依存して調整してもよい。前記治療用組成物を、例えば、同時の又は別の繰り返しのＭＳＣの皮下注射と組み合わせた、接種されたマトリックス又はゲルの局所投与のように、共に投与してもよい。Provided are therapeutic compositions and associated formation methods and uses for improving wound healing. The therapeutic composition comprises an applied amount of MSC inoculated in either a biodegradable matrix, a gel, or a solution suitable for subcutaneous injection. The dosage of the MSC, may range from about 1 × 10 6 MSC~ about 1 × 10 10 per dose. The dosing regime may be daily, weekly, or monthly. The dosage and treatment interval may be adjusted depending on the patient's response to treatment. The therapeutic compositions may be co-administered, for example, topical administration of the inoculated matrix or gel in combination with a simultaneous or another subcutaneous injection of MSC.
Description
関連出願の相互参照
本願は２０１０年４月１日に提出された「創傷治癒を改善する細胞治療」と題する米国特許仮出願第61/341,652号明細書の優先権及び利益を主張し、その全体が参照により本願に組み込まれる。
CROSS REFERENCE TO RELATED APPLICATIONS This application claims priority and benefit of US Provisional Application No. 61 / 341,652 entitled “Cell Therapy to Improve Wound Healing” filed April 1, 2010, which is incorporated herein in its entirety. Is incorporated herein by reference.
創傷治癒は、損傷に続く組織完全性の回復を達成するよう設計された、生化学的及び細胞の事象の高度に制御されたカスケードを含む、複雑な過程である。創傷治癒過程は三つの重複する段階、すなわち炎症段階、増殖段階及び再構築段階に分離され得る。創傷の発生の直後、体の細胞洗浄と一緒に止血の回復を開始する炎症段階と共に治癒が始まる。 Wound healing is a complex process involving a highly controlled cascade of biochemical and cellular events designed to achieve restoration of tissue integrity following injury. The wound healing process can be separated into three overlapping phases: an inflammation phase, a proliferation phase and a remodeling phase. Immediately after the occurrence of the wound, healing begins with an inflammatory phase that begins to restore hemostasis along with cell washing of the body.
炎症段階において、損傷領域は血漿、細胞及び血小板を含む細胞成分で満たされ、フィブリンに富む防壁（血餅）の凝固及び形成を促進して創傷を塞ぎ、創傷部位で微生物の侵入に対して防御する。血小板は脱顆粒を受け、血小板由来成長因子（ＰＤＧＦ）、形質転換成長因子ベータ（ＴＧＦ−β）、表皮成長因子（ＥＧＦ）、形質転換成長因子アルファ（ＴＧＦ−α）、血管内皮成長因子（ＶＥＧＦ）、及び粘着性糖タンパク質を含む多数の治癒介在物質を分泌する。血管作用因子及び化学走化性因子もまた凝固の間に放出され、好中球及び単球のような炎症細胞を創傷へリクルートする。創傷で現われる最初の細胞は好中球であり、次に４８〜７２時間後にマクロファージが創傷部位に存在する支配的な細胞となる。 During the inflammatory phase, the damaged area is filled with cellular components including plasma, cells and platelets, promoting the coagulation and formation of a fibrin-rich barrier (clot) to close the wound and protect against microbial invasion at the wound site To do. Platelets undergo degranulation, platelet-derived growth factor (PDGF), transforming growth factor beta (TGF-β), epidermal growth factor (EGF), transforming growth factor alpha (TGF-α), vascular endothelial growth factor (VEGF). ), And secretes numerous healing mediators including adhesive glycoproteins. Vasoactive and chemotactic factors are also released during coagulation, recruiting inflammatory cells such as neutrophils and monocytes to the wound. The first cells that appear in the wound are neutrophils, then macrophages become the dominant cells present at the wound site 48-72 hours later.
炎症細胞は、創傷部位において細菌、細胞断片、及び外来の抗原を貪食する。更に、炎症細胞は成長因子を産生して線維芽細胞及び内皮細胞をリクルートし、増殖段階のために創傷を準備する。血小板由来因子及び食作用はマクロファージへの変換のための単球活性化を容易にする。活性化したマクロファージは創傷部位の組織修復を制御し、化学走化性因子を放出して血管拡張のために炎症細胞及びプロスタグランジンを誘引する。具体的に、活性化したマクロファージはＰＣＧＦ、ＴＧＦ−α、及びＶＥＧＦを含む多くの成長因子を産生及び分泌して治癒を刺激する。血管拡張及び増大する毛細血管透過性によって、促進する治癒においてヒスタミン、キニン、プロスタグランジン、ロイコトリエン、及び内皮細胞産物が重要な役割を果たす局所浮腫となる。創傷発生から約５〜７日後、線維芽細胞が創傷部位で支配的な細胞型になる。 Inflammatory cells engulf bacteria, cell fragments, and foreign antigens at the wound site. In addition, inflammatory cells produce growth factors to recruit fibroblasts and endothelial cells and prepare the wound for the proliferative stage. Platelet-derived factors and phagocytosis facilitate monocyte activation for conversion to macrophages. Activated macrophages regulate tissue repair at the wound site and release chemotactic factors to attract inflammatory cells and prostaglandins for vasodilation. Specifically, activated macrophages produce and secrete many growth factors including PCGF, TGF-α, and VEGF to stimulate healing. Vasodilation and increased capillary permeability result in local edema where histamine, kinin, prostaglandins, leukotrienes, and endothelial cell products play an important role in promoting healing. Approximately 5-7 days after wounding, fibroblasts become the dominant cell type at the wound site.
炎症段階と重複して、創傷を閉じるために創傷部位において線維芽細胞、内皮細胞、及び上皮細胞の移行と共に増殖段階が始まる。この段階は上皮形成、繊維増殖、及び血管新生を含み、それらの全ては肉芽組織の形成に直接又は間接に寄与する。肉芽組織は、血餅のフィブリン組織に代わる一時的な結合組織として働き、創傷を満たす。この段階の初期は、血管透過性によりタンパク質、サイトカイン、及び細胞成分が漏れ、創傷部位において内皮細胞の増殖を補助する。血管新生が起こり、新たに形成された血管が肉芽組織の形成を補助し、治癒組織に栄養素及び酸素を提供する。内皮細胞の移動及び血管新生は、肉芽組織及び基底内皮膜における細胞外マトリックスの産生及び組織化に加えて、細胞の存在及びサイトカインの発現に依存する。細胞外マトリックスは成長因子の放出を調節し、治癒を補助する。上皮細胞は創傷部位に移動し、増殖して創傷が閉じるまで皮膚を成長させ、再構築段階に入る。 Overlapping with the inflammation phase, the proliferative phase begins with the migration of fibroblasts, endothelial cells, and epithelial cells at the wound site to close the wound. This stage includes epithelialization, fiber growth, and angiogenesis, all of which contribute directly or indirectly to the formation of granulation tissue. The granulation tissue serves as a temporary connective tissue that replaces the fibrin tissue of the clot and fills the wound. Early in this phase, vascular permeability leaks proteins, cytokines, and cellular components, helping endothelial cells grow at the wound site. Angiogenesis occurs, and the newly formed blood vessels assist in the formation of granulation tissue and provide nutrients and oxygen to the healing tissue. Endothelial cell migration and angiogenesis depend on the presence of cells and the expression of cytokines in addition to the production and organization of extracellular matrix in granulation tissue and the basal endothelium. The extracellular matrix regulates growth factor release and aids healing. The epithelial cells migrate to the wound site, proliferate and grow the skin until the wound closes, and enter the remodeling phase.
再構築段階の間、創傷部位の組織は再構成され、典型的な組織構造を取り戻す。コラーゲンは分解酵素によって頻繁に取替え及び作り直しがなされる。細胞外マトリックスは最終的なマトリックスへと成熟し、線維芽細胞は筋線維芽細胞へと変化して収縮性組織として機能する。この最終段階の結果として、血管、線維芽細胞、及び炎症細胞は、移動、アポトーシス、又は他の機構によって創傷部位から消失する。 During the reconstruction phase, the tissue at the wound site is reconstituted to regain typical tissue structure. Collagen is frequently replaced and remade by degrading enzymes. The extracellular matrix matures into the final matrix, and the fibroblasts turn into myofibroblasts and function as contractile tissue. As a result of this final stage, blood vessels, fibroblasts, and inflammatory cells disappear from the wound site by migration, apoptosis, or other mechanisms.
数多くの中で、加齢、栄養失調、脱水症状、感染症、貧血、糖尿病、様々な程度の火傷、慢性腎臓病、及び薬剤のような多くのヒトの状態、病気、治療、及び疾患は、創傷治癒の間、事象の複雑なカスケードを乱す。これらの状態、病気、治療、及び疾患は、治癒過程の炎症段階において創傷治癒を長引かせ又は停止させ、正常な凝固機構を乱し、正常な白血球の機能を乱し、又は血管形成若しくは肉芽組織の形成を妨げる。結果として、患者は痛み、圧痛、腫れ、発熱、治癒遅延、創傷治癒組織（肉芽組織）の脆弱性、におい、創傷破壊、感染症、潰瘍（静脈性、動脈性、糖尿病、又は圧迫）、又はあらゆる形態の長引く創傷を体験し得る。創傷治癒の問題による患者の苦痛は、治療のための高い費用、長い治療期間、不便さ、及び関連する精神的影響によって悪化し得る。 Among many human conditions, illnesses, treatments and diseases such as aging, malnutrition, dehydration, infection, anemia, diabetes, various degrees of burns, chronic kidney disease, and drugs are During wound healing, it disrupts the complex cascade of events. These conditions, illnesses, treatments, and disorders prolong or stop wound healing in the inflammatory stage of the healing process, disrupt normal clotting mechanisms, disrupt normal leukocyte function, or angiogenesis or granulation tissue Prevents the formation of. As a result, the patient has pain, tenderness, swelling, fever, delayed healing, fragility of wound healing tissue (granulation tissue), odor, wound destruction, infection, ulcer (venous, arterial, diabetes, or compression), or You can experience all forms of protracted wounds. Patient suffering from wound healing problems can be exacerbated by high costs for treatment, long treatment periods, inconvenience, and associated mental effects.
１００を超える既知の生理学的要素が、糖尿病を持つ個体の創傷治癒欠損に寄与する。これらは、中でも、減少された又は損なわれた成長因子の産生、血管新生反応、マクロファージ機能、コラーゲンの蓄積、表皮の防壁機能、肉芽組織の量、並びにケラチノサイト及び線維芽細胞の移動及び増殖を含む。例えば、糖尿病患者の足の創傷はデフィニション（definition）によってしばしば慢性的となり、減少された血管新生反応、神経障害、外傷、及び足変形という結果になり得る。 More than 100 known physiological factors contribute to wound healing deficits in individuals with diabetes. These include, among other things, reduced or impaired growth factor production, angiogenic responses, macrophage function, collagen accumulation, epidermal barrier function, granulation tissue volume, and keratinocyte and fibroblast migration and proliferation . For example, diabetic foot wounds often become chronic due to definition and can result in reduced angiogenic response, neuropathy, trauma, and foot deformity.
従って、創傷治癒の領域において病気及び状態に関する前述の問題に対処する治療用組成物（therapeutic composition）及び細胞治療、特に慢性的な創傷の治癒が現在必要とされている。 Accordingly, there is a current need for therapeutic compositions and cell therapies, particularly chronic wound healing, that address the aforementioned problems related to diseases and conditions in the area of wound healing.
本発明は創傷治癒を改善するための治療用組成物並びにその製造方法及び使用を提供する。前記治療用組成物は、治療的に有効な量の細胞を創傷へ送達して治癒を改善するためにビヒクルに接種（seed）された月経由来幹様細胞（stem-like cell）を含む。前記月経由来幹様細胞は月経から単離され、実質的に異種の細胞集団、実質的に同種の細胞集団、又はＣＤ−１１７を発現する細胞集団のいずれか一つ又は組み合わせを含む。 The present invention provides a therapeutic composition for improving wound healing and methods for making and using the same. The therapeutic composition comprises menstrual stem-like cells seeded in a vehicle to deliver a therapeutically effective amount of cells to the wound and improve healing. The menstrual stem-like cells are isolated from menstruation and comprise any one or a combination of a substantially heterogeneous cell population, a substantially homogeneous cell population, or a cell population expressing CD-117.
一つの態様において、前記月経由来幹様細胞は約１×１０6細胞〜約１×１０10細胞の範囲でビヒクルに接種される。別の態様において、治療中、患者の耐性及び創傷治癒の進行に依存して、前記月経由来幹様細胞はビヒクルごとに約１×１０10細胞超で又は約１×１０6細胞未満で接種される。 In one embodiment, the menstrual stem-like cells are seeded in a vehicle in the range of about 1 × 10 6 cells to about 1 × 10 10 cells. In another embodiment, during treatment, the menstrual stem-like cells are inoculated with greater than about 1 × 10 10 cells or less than about 1 × 10 6 cells per vehicle, depending on patient tolerance and progression of wound healing. Is done.
前記ビヒクルは生物分解性マトリックス、ゲル、又は月経由来幹様細胞の皮下注射に適する医薬的に許容し得る賦形剤のいずれか一つであってもよい。 The vehicle may be any one of biodegradable matrices, gels, or pharmaceutically acceptable excipients suitable for subcutaneous injection of menstrually derived stem-like cells.
創傷治癒を改善するための組成物を調製する方法が提供される。前記方法は、創傷に細胞を投与するために治療的に有効な量の月経由来幹様細胞をビヒクルに接種する工程を含む。前記接種工程は担体に約１×１０6細胞〜約１×１０10細胞の範囲で月経由来幹様細胞を加える工程を含む。別の態様において、前記月経由来幹様細胞はビヒクルごとに約１×１０10細胞超で又は約１×１０6細胞未満で接種される。 A method of preparing a composition for improving wound healing is provided. The method includes inoculating the vehicle with a therapeutically effective amount of menstrual stem-like cells to administer the cells to the wound. The inoculation step includes adding menstruation-derived stem-like cells to the carrier in the range of about 1 × 10 6 cells to about 1 × 10 10 cells. In another embodiment, the menstrual stem-like cells are inoculated with more than about 1 × 10 10 cells or less than about 1 × 10 6 cells per vehicle.
前記月経由来幹様細胞は、実質的に異種の細胞集団、実質的に同種の細胞集団、又はＣＤ−１１７を発現する細胞集団のいずれか一つ以上であってもよい。 The menstrual stem-like cells may be any one or more of a substantially heterogeneous cell population, a substantially homogeneous cell population, or a cell population expressing CD-117.
創傷治癒を改善するための方法もまた提供される。前記方法は、適用量のビヒクル中に接種された治療的に有効な量の月経由来幹様細胞を、少なくとも一回創傷へ投与する工程を含む。一つの態様において、前記方法は、月経由来幹様細胞を接種されたビヒクルを創傷へ投与する前に、十分に創傷を清拭する工程を含む。前記適用量の月経由来幹様細胞は、ビヒクルごとに約１×１０6細胞〜約１×１０10細胞の範囲で接種されてもよい。前記適用量の月経由来幹様細胞は、ビヒクルごとに約１×１０10細胞超で又は約１×１０6細胞未満で接種されてもよい。 A method for improving wound healing is also provided. The method comprises the step of administering to the wound at least once a therapeutically effective amount of menstrual stem-like cells inoculated in an applied amount of vehicle. In one embodiment, the method comprises the step of thoroughly wiping the wound prior to administering the vehicle inoculated with menstrual stem-like cells to the wound. The applied amount of menstrual stem-like cells may be seeded in a range of about 1 × 10 6 cells to about 1 × 10 10 cells per vehicle. The applied amount of menstrual stem-like cells may be seeded at greater than about 1 × 10 10 cells or less than about 1 × 10 6 cells per vehicle.
代わりの態様において、前記方法は、適用量（dosage）の月経由来幹様細胞を接種された少なくとも２形態のビヒクルを創傷へ投与する工程を含む。 In an alternative embodiment, the method comprises administering to the wound at least two forms of vehicle inoculated with a dosage of menstrual stem-like cells.
月経から単離された治療的に有効な量の多能性の細胞を接種された生物分解性マトリックス又はゲルは、治療中の患者の耐性及び創傷治癒の進行に依存する１ヶ月から６ヶ月までまたはそれより長い治療期間、日、週又は月に少なくとも一回投与される。 Biodegradable matrix or gel inoculated with a therapeutically effective amount of pluripotent cells isolated from menstruation from 1 month to 6 months depending on the tolerance of the patient being treated and the progress of wound healing Or at least once a longer treatment period, day, week or month.
治療的に有効な適用量の月経由来幹様細胞を接種された医薬的に許容し得る賦形剤もまた、治療中の患者の耐性及び創傷治癒の進行に依存する一ヶ月から六ヶ月までまたはそれより長い治療期間、毎日、毎週、毎月基準（basis）で皮下注射によって創傷へ一緒に投与されてもよい。 Pharmaceutically acceptable excipients inoculated with therapeutically effective dosages of menstrual stem-like cells also vary from 1 month to 6 months depending on the tolerance of the patient being treated and the progress of wound healing Or it may be administered together to the wound by subcutaneous injection on a daily, weekly, monthly basis (basis) for longer treatment periods.
一つの態様において、治療的に有効な量の月経由来幹様細胞を接種された前記医薬的に許容し得る賦形剤は、治療中の患者の耐性及び創傷治癒の進行に依存する１ヶ月から６ヶ月までまたはそれより長い治療期間、毎週皮下注射によって創傷へ投与される。 In one embodiment, the pharmaceutically acceptable excipient inoculated with a therapeutically effective amount of menstrual stem-like cells is one month depending on the tolerance of the patient being treated and the progress of wound healing Administered to the wound by weekly subcutaneous injection for up to 6 months or longer treatment period.
本発明は、創傷治癒を改善するための、月経由来幹様細胞（ＭＳＣ）を用いる治療用組成物並びにその製造方法及び使用を提供する。ＭＳＣは、創傷治癒を改善又は促進する目的及び創傷治癒に関する他の問題に対処する目的のために治療用組成物中で対象に投与されてもよい。創傷部位へのＭＳＣの投与は、ＭＳＣの創傷への体の自然な反応の模倣を誘引し及び／又は引き起こす。例えば、ＭＳＣは自然な創傷治癒機構を含む及び／又は補助する他の細胞型、例えば、線維芽細胞、炎症細胞、又は血管新生のための細胞に分化し、又は化学走化性因子、成長因子、又は他の創傷治癒介在物質を産生及び分泌してもよい。 The present invention provides therapeutic compositions using menstrual stem-like cells (MSCs) and methods for making and using the same to improve wound healing. MSCs may be administered to a subject in a therapeutic composition for purposes of improving or promoting wound healing and addressing other issues related to wound healing. Administration of MSC to the wound site induces and / or causes mimics of the body's natural response to the wound of MSC. For example, MSCs differentiate into other cell types that include and / or assist with natural wound healing mechanisms, such as fibroblasts, inflammatory cells, or cells for angiogenesis, or chemotactic factors, growth factors Or other wound healing mediators may be produced and secreted.
月経由来幹様細胞
用語「月経由来幹様細胞」、「ＭＳＣ」は、参照によりその全体が本願に組み込まれる米国特許出願第12/615,372号明細書の方法に従って月経から単離される、あらゆる生存可能な高増殖性の多能性細胞（multipotent cell）集団、さもなければ多能性細胞（pluripotent cell）集団を意味し、前記集団は、異種の細胞集団又は、同種の細胞集団、系統中立の（lineage-uncomitted）細胞集団、系統コミットな（lineage-committed）細胞集団、表面マーカーＣＤ１１７及び／又はＳＳＥＡ−４を積極的に発現する細胞集団、又はそれから派生するあらゆる細胞集団を含むがそれらに限定されない、それから派生するあらゆる細胞集団である。
The term “menstrual stem-like cell”, “MSC” is any term isolated from menstruation according to the method of US patent application Ser. No. 12 / 615,372, which is incorporated herein by reference in its entirety. Means a viable, highly proliferative multipotent cell population, otherwise a pluripotent cell population, said population being a heterogeneous cell population, a homogeneous cell population, a lineage neutral A lineage-uncomitted cell population, a lineage-committed cell population, a cell population that actively expresses the surface markers CD117 and / or SSEA-4, or any cell population derived therefrom. Any cell population derived therefrom without limitation.
発明を実施するための月経由来幹様細胞は、本開示において提示される機能及び方法を実行できる十分な量のＭＳＣを生産する、いかなる適切な技術を使用して単離されてもよい。これらの方法の例は米国特許出願第12/615,372号明細書において記載されている方法を含むがそれらに限定されない。細胞表面マーカーはＭＳＣを単離する及び／又は豊富にするための別の手段を提供する。例えば、ＭＳＣ上のＣＤ１１７のような表面マーカーは、米国特許出願第12/615,372号明細書において記載されている特定のモノクローナル又はポリクローナル抗体と反応性である。抗体はＭＳＣ細胞集団を捕獲し又は豊富にするための試薬として使用され得る。 Menstrual stem-like cells for practicing the invention may be isolated using any suitable technique that produces sufficient amounts of MSCs to perform the functions and methods presented in this disclosure. Examples of these methods include, but are not limited to, those described in US patent application Ser. No. 12 / 615,372. Cell surface markers provide another means to isolate and / or enrich MSCs. For example, surface markers such as CD117 on MSC are reactive with certain monoclonal or polyclonal antibodies described in US patent application Ser. No. 12 / 615,372. The antibody can be used as a reagent to capture or enrich MSC cell populations.
ＭＳＣ単離
非制限的な態様として、ＭＳＣは月経から単離され、抗生物質処理で洗われ、回収されてもよい。ＭＳＣは（ｉ）細胞の新鮮な供給源、（ｉｉ）細胞の新鮮な供給源の培養物、（ｉｉｉ）新鮮な細胞の培養物からの細胞単離株（すなわち、ＣＤ１１７選択又は他の積極的若しくは消極的選択によって得られる）、（ｉｖ）凍結保存後に解凍された細胞、（ｖ）冷凍保存され、次に解凍された後に培養される細胞、（ｖｉ）凍結保存されて解凍された試料の培養物からの細胞単離株、又は（ｖｉｉ）米国特許出願第12/615,372号明細書において記載されているような他のあらゆる細胞集団として使用され、本発明の治療用組成物に接種してもよい。
MSC isolation As a non-limiting embodiment, MSCs may be isolated from menstruation, washed with antibiotic treatment and recovered. MSCs are (i) a fresh source of cells, (ii) a fresh source culture of cells, (iii) a cell isolate from a fresh cell culture (ie, CD117 selection or other active Or (iv) obtained by passive selection), (iv) cells thawed after cryopreservation, (v) cells that are cryopreserved and then cultured after thawing, (vi) samples that have been cryopreserved and thawed A cell isolate from culture, or (vii) used as any other cell population as described in US patent application Ser. No. 12 / 615,372, inoculated with the therapeutic composition of the present invention. Also good.
一つの非制限的な例において、ＭＳＣは、約１〜３時間回収カップを膣内に配置して回収した月経から単離される。二つの別個の回収物から回収された月経を、下記のように冷却された培地を含む二つの回収チューブに入れる：第一のチューブはヘパリンナトリウムを含むＨＢＳＳ及び抗生物質を含む培地を有し、第二のチューブはヘパリンナトリウムを含むＨＢＳＳ及び、例えば、米国特許出願第12/615,372号明細書において記載されているような抗生物質を含む培地を有する。 In one non-limiting example, MSCs are isolated from menstruation collected by placing a collection cup in the vagina for about 1-3 hours. Menstruations collected from two separate collections are placed in two collection tubes containing chilled media as follows: the first tube has HBSS containing sodium heparin and a medium containing antibiotics; The second tube has a medium containing HBSS containing sodium heparin and antibiotics as described, for example, in US patent application Ser. No. 12 / 615,372.
培地及び月経を含む前記回収チューブは密封され、約２４〜約７２時間以内の回収工程のために、冷凍形態のレンガを有するスタイロフォーム冷却器に輸送される。培地試料内の前記月経は抗生物質カクテル中で抗生物質洗浄を受ける。最終濃縮カクテルはＨＢＳＳ中にバンコマイシン（１６０μｇ／ｍｌ）、クラフォラン（５００μｇ／ｍｌ）、アミカシン（２００μｇ／ｍｌ）、ゲンタマイシン（２４０μｇ／ｍｌ）、及びアンフォテリシンＢ（５．４μｇ／ｍｌ）を含み、これは回収に用いられる濃度と同じである。 The collection tube containing medium and menstruation is sealed and transported to a styrofoam cooler with bricks in frozen form for a collection process within about 24 to about 72 hours. The menstruation in the medium sample undergoes antibiotic washing in an antibiotic cocktail. The final concentrated cocktail contains vancomycin (160 μg / ml), claforan (500 μg / ml), amikacin (200 μg / ml), gentamicin (240 μg / ml), and amphotericin B (5.4 μg / ml) in HBSS, It is the same concentration used for recovery.
第一のチューブにおいて、１ｍｌの原液抗生物質カクテルを１９ｍｌのＨＢＳＳ及び０．４ｍｌのヘパリン（４００ユニット）に加える。第二のチューブにおいて、０．２ｍｌのヘパリン（２００ユニット）を１０ｍｌのＤＰＢＳに加える。 In the first tube, add 1 ml stock antibiotic cocktail to 19 ml HBSS and 0.4 ml heparin (400 units). In a second tube, add 0.2 ml heparin (200 units) to 10 ml DPBS.
第一のチューブ（ヘパリン及びＤＰＢＳ又は代わりにＨＢＳＳ）内の月経試料は抗生物質洗浄処理を受ける。前記試料は１００ミクロンフィルターを用いて５０ｍｌ円錐管に濾過される。第一の回収チューブを１０ｍｌのＤＰＢＳ及び０．２ｍｌのヘパリンで洗浄し、５０ｍｌ円錐管に加える。次に前記５０ｍｌ円錐管を８４０ｇ、７分間、４．０℃で遠心分離する。上清はペレットを乱さないよう注意して除去される。１ｍｌの希釈抗生物質カクテル及び０．４ｍｌのヘパリンをＨＢＳＳと共に残ったペレットに加え、ペレットを再懸濁する。細胞を含むこの混合物を２〜８℃で２０〜３０時間培養し、培養の初めの５時間は試料を回転装置に置く。培養の後、細胞の混合物を同じ遠心分離パラメーターで再び遠心分離し、ペレットを５ｍｌのＤＰＢＳ又はＨＢＳＳに再懸濁してもよい。 Menstrual samples in the first tube (heparin and DPBS or alternatively HBSS) are subjected to an antibiotic wash treatment. The sample is filtered into a 50 ml conical tube using a 100 micron filter. The first collection tube is washed with 10 ml DPBS and 0.2 ml heparin and added to a 50 ml conical tube. The 50 ml conical tube is then centrifuged at 840 g for 7 minutes at 4.0 ° C. The supernatant is removed with care not to disturb the pellet. Add 1 ml of diluted antibiotic cocktail and 0.4 ml of heparin to the remaining pellet with HBSS and resuspend the pellet. This mixture containing cells is incubated at 2-8 ° C. for 20-30 hours, and the sample is placed on a rotator for the first 5 hours of culture. After incubation, the cell mixture may be centrifuged again with the same centrifugation parameters and the pellet resuspended in 5 ml DPBS or HBSS.
第二のチューブ（ヘパリン、ＨＢＳＳ）内の前記月経試料を感染症試験に用いる。 The menstrual sample in the second tube (heparin, HBSS) is used for infectious disease testing.
細胞数、細胞生存率、感染症試験、及び微生物学的試験を、米国特許出願第12/615,372号明細書に記載されている技術に従って、二つの月経細胞試料のそれぞれで行ってよい。 Cell number, cell viability, infection testing, and microbiological testing may be performed on each of the two menstrual cell samples according to the techniques described in US patent application Ser. No. 12 / 615,372.
次に、前記二つの月経細胞試料（約５ｍｌ）を、米国特許出願第12/615,372号明細書に記載されている技術に従って、凍結保存してもよい。５ｍｌの凍結保存培地（ＤＰＢＳ中に２０％のＨＳＡ及び２０％のＤＭＳＯ）を二つの月経細胞試料のそれぞれに個別に加える。 The two menstrual cell samples (about 5 ml) may then be cryopreserved according to the technique described in US patent application Ser. No. 12 / 615,372. 5 ml of cryopreservation medium (20% HSA and 20% DMSO in DPBS) is added individually to each of the two menstrual cell samples.
凍結保存細胞を、米国特許出願第12/615,372号明細書に記載されている技術に従って、解凍してもよい。代わりに、それぞれの細胞試料を更に処理して、特定の細胞マーカー、例えばＣＤ１１７、を発現する細胞を単離してもよい。 Cryopreserved cells may be thawed according to the techniques described in US patent application Ser. No. 12 / 615,372. Alternatively, each cell sample may be further processed to isolate cells that express a particular cell marker, such as CD117.
細胞選択を、米国特許出願第12/615,372号明細書に開示されている技術に従って、２５０万の細胞から１億までの細胞について実施してもよい。 Cell selection may be performed on 2.5 to 100 million cells according to the technique disclosed in US patent application Ser. No. 12 / 615,372.
細胞選択の前に、新鮮な細胞又は解凍した細胞を氷上に少なくとも１５分間置く。細胞を３００ｇ、１０分間、４℃で遠心分離する。上清を除去し、ペレットを３００ｍｌのワーキングバッファー（working buffer）に、ワーキングバッファー１０ｍｌにつき約１滴のＤＮＡ分解酵素パルモザイム、１００μｌのＦｅＲブロッキング試薬、及び１００μｌのＣＤ１１７ミクロビーズと共に再懸濁する（米国特許出願第12/615,372号明細書に記載されているように）。この混合物を２０〜２５分間、氷上で冷やす。２ｍｌのワーキングバッファーを加える。前記混合物を含むチューブを１０分間、３００ｇ、４℃で遠心分離する。上清を除去し、ペレットを５００μｌのワーキングバッファーに再懸濁する。 Place fresh or thawed cells on ice for at least 15 minutes prior to cell selection. The cells are centrifuged at 300 g for 10 minutes at 4 ° C. The supernatant is removed and the pellet is resuspended in 300 ml working buffer with about 1 drop of DNase palmozyme, 100 μl FeR blocking reagent, and 100 μl CD117 microbeads per 10 ml working buffer (USA) As described in patent application 12 / 615,372). The mixture is chilled on ice for 20-25 minutes. Add 2 ml working buffer. The tube containing the mixture is centrifuged for 10 minutes at 300 g at 4 ° C. The supernatant is removed and the pellet is resuspended in 500 μl working buffer.
次に、前記再懸濁した細胞を磁石台上でＭＳカラム（約５００μｌのワーキングバッファーで湿らせる）に通す。前記ＭＳカラムを５００μｌのワーキングバッファーで３回洗浄する。洗浄されてカラムを通る非ラベル細胞（non-labeled cell）を取り出し、細胞数、細胞生存率、等を分析する。次に、前記ＭＳカラムを磁石台から移して、１ｍｌのワーキングバッファーをＭＳカラムに通し、ラベル細胞（labeled cell）を取り出す。回収されたラベル細胞を別のＭＳカラムに通してラベル細胞を濃縮してもよい。この場合、ラベル細胞はＣＤ１１７細胞である。 The resuspended cells are then passed through an MS column (wet with about 500 μl of working buffer) on a magnetic platform. The MS column is washed 3 times with 500 μl working buffer. Non-labeled cells that have been washed and pass through the column are removed and analyzed for cell number, cell viability, and the like. Next, the MS column is moved from the magnet stand, 1 ml of working buffer is passed through the MS column, and labeled cells are taken out. The collected labeled cells may be passed through another MS column to concentrate the labeled cells. In this case, the label cell is a CD117 cell.
前述の処理は普遍的予防策を取って生物学的安全キャビネットにおける無菌操作に従って実施する。 The aforementioned treatment is carried out according to aseptic operation in a biological safety cabinet taking universal precautions.
次に、単離したＣＤ１１７＋ＭＳＣを、米国特許出願第12/615,372号明細書に開示されている技術に従って、冷凍保存してもよい。次に、冷凍保存した又は新鮮な細胞を本発明に従ってマトリックス、ゲル、又は医薬的に許容し得る賦形剤に接種するために調製する。ＣＤ１１７選択細胞を解凍する（凍結保存しているならば）。細胞の使用の前に、試料を５回洗浄して細胞から抗生物質を除去する。前記細胞を１０ｍｌのＤＰＢＳ及び０．２ｍｌのヘパリン（防腐剤なし）で洗浄する。試料を軽くボルテックスし、８４０ｇ、７分間、４．０℃で遠心分離する。上清を除去し、ペレットを５回洗い直して、処理用の細胞を準備する。次に、解凍した又は新鮮なＣＤ１１７細胞を、増殖のために１５％のチャン完全培地（Chan's complete media）又はヒト血清ＤＭＥＭ完全培地（又は等価物）に蒔く。 The isolated CD117 + MSC may then be stored frozen according to the techniques disclosed in US patent application Ser. No. 12 / 615,372. The cryopreserved or fresh cells are then prepared for inoculation into a matrix, gel, or pharmaceutically acceptable excipient according to the present invention. Thaw CD117 selected cells (if cryopreserved). Prior to use of the cells, the sample is washed 5 times to remove antibiotics from the cells. The cells are washed with 10 ml DPBS and 0.2 ml heparin (no preservative). Lightly vortex the sample and centrifuge at 840 g for 7 minutes at 4.0 ° C. The supernatant is removed and the pellet is rewashed 5 times to prepare the cells for processing. The thawed or fresh CD117 cells are then plated on 15% Chan's complete media or human serum DMEM complete medium (or equivalent) for growth.
十分な増殖の後、細胞を任意にＣＤ１１７に関して選択し、再び増殖させてもよい。ＣＤ１１７選択細胞の一回の増殖又は二回の増殖の目的は、応用のために所望の数の細胞を得ることである。接種のために、これらの増殖したＣＤ１１７＋細胞をすぐに使用してもよいし、又は凍結保存して、後に解凍してもよい。 After sufficient growth, the cells may optionally be selected for CD117 and expanded again. The purpose of single or double expansion of CD117 selected cells is to obtain the desired number of cells for the application. These seeded CD117 + cells may be used immediately for inoculation or may be stored frozen and later thawed.
製剤
本発明は、ＭＳＣ及び医薬的に許容し得る賦形剤又はマトリックス又はゲルを含む治療用組成物を提供する。対象の治療用組成物は、混合物中に医薬的に許容し得る賦形剤又はマトリックス又はゲル及びＭＳＣを単独で又は１以上の生物学的活性物質（例えば、成長因子のような）の組み合わせにおいて、治癒を改善するための様々な方法による患者の創傷への投与に効果的な濃度（strength）で含む。生物学的活性物質を含む治療用組成物のあらゆる調製物は、本発明の代替的な特徴である。更に、治療に望ましいのであれば、前記組成物は湿潤剤又は乳化剤のような少量の補助物質、又はＭＳＣの効果を高めるｐＨ緩衝剤を含んでもよい。
Formulations The present invention provides a therapeutic composition comprising MSC and a pharmaceutically acceptable excipient or matrix or gel. The subject therapeutic composition comprises a pharmaceutically acceptable excipient or matrix or gel and MSC in a mixture alone or in combination of one or more biologically active agents (such as growth factors). Including at effective concentrations for administration to a patient's wound by various methods to improve healing. Any preparation of a therapeutic composition comprising a biologically active substance is an alternative feature of the present invention. In addition, if desired for treatment, the composition may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, or pH buffering agents that enhance the effectiveness of MSCs.
ＭＳＣが組み込まれ又は埋め込まれ得るマトリックスは、生物学的適合性、レシピエント適合性のマトリックス、及びレシピエントに有害でない産物へ分解するマトリックスを含む。これらのマトリックスはＭＳＣのための補助及び保護を提供する。非制限的な例として、ＭＳＣを接種されたバイオマトリックスを調製するために、以下のマトリックスのいずれもが使用されてよい：インテグラ二層マトリックス創傷被覆材（Integra Bilayer Matrix Wound Dressing）、プロメガマトリックス創傷被覆材（Promega Matrix Wound Dressing）、又は等価物。一つの態様において、創傷被覆材全体を使用してもよく、又はより小さい断片に切断してもよい。別の態様において、４つの等しい断片を使用してもよい。所望量のＭＳＣを接種するために、二つのＴ７５フラスコ及び二つのシャーレの中に前記創傷被覆材を置く。ＭＳＣを１５％チャン培地（３２５ｍｌのＭＥＭα、７５ｍｌのＦＢＳ、９０ｍｌのＣｈａｎｇ Ｂ、１０ｍｌのＣｈａｎｇ Ｃ、５ｍｌのＰｅｎｓｔｒｅｐ、及び５ｍｌのＬ−グルタミン）中に蒔いてもよい。細胞量は選択される。次に、チャン培地及び所望量のＭＳＣの混合物を、選択したマトリックスにゆっくりと適用することにより、ＭＳＣをマトリックス中に接種する。具体的には、細胞が材料上に吸収されるように、自動ペレットから直接マトリックス上にゆっくりと放出することにより、ＭＳＣを接種した。 Matrix in which MSCs can be incorporated or embedded include biocompatible, recipient compatible matrices, and matrices that degrade into products that are not harmful to the recipient. These matrices provide assistance and protection for the MSC. As a non-limiting example, any of the following matrices may be used to prepare MSC inoculated biomatrix: Integra Bilayer Matrix Wound Dressing, Promega Matrix Wound Dressing (Promega Matrix Wound Dressing) or equivalent. In one embodiment, the entire wound dressing may be used or cut into smaller pieces. In another embodiment, four equal fragments may be used. Place the wound dressing in two T75 flasks and two petri dishes to inoculate the desired amount of MSC. MSCs may be sown in 15% Chang's medium (325 ml MEMα, 75 ml FBS, 90 ml Chang B, 10 ml Chang C, 5 ml Penstrep, and 5 ml L-glutamine). The amount of cells is selected. The MSC is then inoculated into the matrix by slowly applying a mixture of Chang's medium and the desired amount of MSC to the selected matrix. Specifically, MSCs were inoculated by slowly releasing directly from the automated pellets onto the matrix so that the cells were absorbed onto the material.
ＭＳＣをマトリックスに接種する工程の非制限的な例
一つの態様において、マトリックス中の単層のＣＤ１１７＋ＭＳＣ細胞系を培養する方法を提供する。
Non-limiting example of inoculating a matrix with MSC In one embodiment, a method of culturing a monolayer of CD117 + MSC cell line in a matrix is provided.
ＭＳＣ（Ｍ２９１５６ＲＡ ＣＤ１１７陽性画分）の冷凍保存した試料を３７〜４０℃の水槽中で、完全に解凍しきらないように解凍する。部分的に解凍した細胞をＤＮＡ分解酵素（１０ｍｌ毎に１滴）を含む冷やした１５％チャン細胞培地に移し、転倒により穏やかに混合する。５ｍｌの細胞調製物に対し、２５ｍｌの冷やした培地を用いる。調製物を１２０ｇで５分間遠心分離する。上清をピペットで除き、ＤＮＡ分解酵素を含まない１５％完全チャン培地中に転倒によって再懸濁する。再び、１２０ｇで５分間遠心分離する。上清をピペットで除き、１ｃｍ2毎に５０００細胞で細胞をフラスコ中に蒔いて、翌日回収する（例えば、１Ｔ１７５フラスコ中に１００万細胞）；１ｃｍ2毎に１０００細胞で蒔いて、３〜５日中に回収する。 A frozen sample of MSC (M29156RA CD117 positive fraction) is thawed in a 37-40 ° C. water bath so that it is not completely thawed. Partially thawed cells are transferred to chilled 15% Chang cell medium containing DNA-degrading enzyme (1 drop per 10 ml) and gently mixed by inversion. Use 25 ml of chilled medium for 5 ml of cell preparation. The preparation is centrifuged at 120 g for 5 minutes. The supernatant is pipetted off and resuspended by inversion in 15% complete Chang's medium without DNase. Again centrifuge at 120 g for 5 minutes. Remove the supernatant with a pipette and seed cells in a flask with 5000 cells every 1 cm 2 and collect the next day (eg 1 million cells in a 1T175 flask); seed with 1000 cells every 1 cm 2 and 3-5 Collect during the day.
７５〜８０％コンフルエントになると、細胞をトリプシン処理できる。トリプシン処理の前に、適当量のＴｒｙｐＬＥ（トリプシン様酵素）、チャン培地、及びＤＰＢＳを約３６．０〜３８．０℃で温める。培地をフラスコから吸引する。Ｔ−２５、Ｔ−７５又はＴ−１７５フラスコを５，１０及び２５ｍｌのカルシウム及びマグネシウムを含まない予熱したＤＰＢＳでそれぞれ洗浄する。Ｔ−２５、Ｔ−７５又はＴ−１７５に１．５ｍｌ、３ｍｌ及び６ｍｌの予熱したＴｒｙｐＬＥをそれぞれ加える。ＣＯ2培養器において、３６．０〜３８．０℃で約５分間、フラスコを攪拌及びインキュベートする。穏やかに細胞を取り除き、フラスコの内容物を、前に加えたＴｒｙｐＬＥと同じ量のチャン培地で希釈する。フラスコの内容物を５０ｍｌ遠心分離チューブに移す。Ｔ−２５、Ｔ−７５又はＴ−１７５非組織培養処理（non-tissue culture treated）フラスコを追加の５、１０及び２５ｍｌのカルシウム又はマグネシウムを含まないＤＰＢＳでそれぞれ洗浄する。洗浄液をフラスコの内容物を含む５０ｍｌ遠心分離チューブに加える。前記チューブを１２０ｇで５分間遠心分離する。遠心分離後、上清を廃棄する。細胞ペレットの体積を記録し、２０μｌを取り出して細胞数を数える。 When 75-80% confluent, cells can be trypsinized. Prior to trypsinization, warm appropriate amounts of TrypLE (trypsin-like enzyme), Chang's medium, and DPBS at about 36.0-38.0 ° C. Aspirate the medium from the flask. Wash T-25, T-75 or T-175 flasks with 5,10 and 25 ml preheated DPBS without calcium and magnesium, respectively. Add 1.5 ml, 3 ml and 6 ml of preheated TrypLE to T-25, T-75 or T-175, respectively. In a CO 2 incubator, stir and incubate the flask at 36.0-38.0 ° C. for about 5 minutes. Gently remove the cells and dilute the flask contents with the same amount of Chang's medium as TrypLE added previously. Transfer the contents of the flask to a 50 ml centrifuge tube. Wash the T-25, T-75 or T-175 non-tissue culture treated flasks with an additional 5, 10 and 25 ml of DPBS without calcium or magnesium, respectively. Add wash to 50 ml centrifuge tube containing flask contents. The tube is centrifuged at 120 g for 5 minutes. After centrifugation, discard the supernatant. Record the volume of the cell pellet, remove 20 μl and count the number of cells.
マトリックスに接種する工程を、まず殺菌したハサミを用いてマトリックスを所望の大きさに切断して適切な大きさのシャーレの中に置いて、実施する。培地を前記シャーレに加え、マトリックスに吸収させる。２００万の細胞（又は他の細胞量）をマトリックス上に、中心から始めて端へと渦状にゆっくりと蒔く。細胞をマトリックス中に安定させる。所望の大きさのマトリックスを打ち抜き、細胞側が創傷へ向くように創傷へ移す。残りのＭＳＣを別のマトリックス上に蒔いてもよく、又は米国特許出願第12/615,372号明細書に開示されている方法に従って濃縮及び冷凍保存してもよい。 The step of inoculating the matrix is performed by first cutting the matrix into a desired size using sterilized scissors and placing it in a petri dish of an appropriate size. The medium is added to the petri dish and absorbed into the matrix. Slowly vortex 2 million cells (or other amounts of cells) onto the matrix, starting from the center and ending in an edge. Stabilize cells in matrix. The matrix of the desired size is punched and transferred to the wound with the cell side facing the wound. The remaining MSCs may be spread on a separate matrix or may be concentrated and stored frozen according to the methods disclosed in US patent application Ser. No. 12 / 615,372.
別の態様において、ＭＳＣの成長及び／又は分化を補助する生体吸収性ゲルを含む混合物にＭＳＣを混合してもよい。細胞量は選択される。次に、チャン培地及び所望量のＭＳＣの混合物をゲルへゆっくりと適用することにより、ＭＳＣをゲル中に穏やかに接種する。具体的には、細胞がゲル中に穏やかに混合されるように自動ピペットから直接ゲル上へゆっくりとＭＳＣを放出して、ＭＳＣを接種してもよい。非制限的な例として、ダーマサイエンス社が製造する、ダーマグラン−Ｂ ３Ｏｚ創傷ゲル（チューブ）、ダーマグラン−Ｂ親水性創傷ゲルがある。他の適切なゲルは、慢性又は急性の創傷用の主要な被覆又は充填剤のいずれかを提供する、親水性の包帯剤を含んでもよい。ゲルは、創傷治癒に繋がる湿性、少し酸性の環境をビタミンＡ、ビタミンＢ６、カルシウム及びマグネシウムと共に提供する、亜鉛−栄養素包帯剤及び平衡ｐＨ技術（balanced pH technology）を含んでもよい。 In another embodiment, MSCs may be mixed in a mixture comprising a bioabsorbable gel that assists MSC growth and / or differentiation. The amount of cells is selected. The MSC is then gently inoculated into the gel by slowly applying a mixture of Chang's medium and the desired amount of MSC to the gel. Specifically, MSCs may be inoculated by slowly releasing MSCs directly from the automatic pipette onto the gel so that the cells are gently mixed into the gel. Non-limiting examples include Dermaglan-B 3Oz wound gel (tube), Dermaglan-B hydrophilic wound gel manufactured by Derma Science. Other suitable gels may include hydrophilic dressings that provide either the primary coating or filler for chronic or acute wounds. The gel may include a zinc-nutrient dressing and a balanced pH technology that provides a moist, slightly acidic environment with vitamin A, vitamin B6, calcium and magnesium leading to wound healing.
ＭＳＣをまた、装置、例えばシリンジ、による皮下注射での送達のために、医薬的に許容し得る担体又は賦形剤と混合してもよい。本願に記載される前記医薬的に許容し得る担体又は賦形剤、例えばビヒクル、補助剤、担体又は希釈剤、は、一般に容易に入手可能である。一般に、医薬的に許容し得る担体又は賦形剤は、細胞に対して化学的に不活性なものであり、且つ、使用条件下で有害な副作用又は毒性を有さないものである。例は、シラン、溶媒、分散媒、細胞培養培地、リン酸塩、クエン酸塩のような水性緩衝液、及び他の有機酸；アスコルビン酸を含む酸化防止剤；低分子量（約１０残基未満）ポリペプチド；血清アルブミン、ゼラチン、又は免疫グロブリンのようなタンパク質；ポリビニルピロリドンのような親水性ポリマー；グリシン、グルタミン、アスパラギン、アルギニン又はリシンのようなアミノ酸；単糖、二糖、及びグルコース、マンノース、又はデキストリンを含む他の炭水化物；ＥＤＴＡのようなキレート剤；マンニトール又はソルビトールのような糖アルコール；ナトリウムのような塩形成対イオン；及び／又は非イオン性界面活性剤を含むが、それらに制限されない。あらゆる担体又は賦形剤は、殺菌され、シリンジを介して容易に送達できる程度に流動性である。 The MSC may also be mixed with a pharmaceutically acceptable carrier or excipient for delivery by subcutaneous injection through a device, such as a syringe. The pharmaceutically acceptable carriers or excipients described herein are generally readily available, such as vehicles, adjuvants, carriers or diluents. In general, pharmaceutically acceptable carriers or excipients are those that are chemically inert to the cells and that do not have deleterious side effects or toxicity under the conditions of use. Examples are silanes, solvents, dispersion media, cell culture media, aqueous buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) ) Polypeptides; proteins such as serum albumin, gelatin or immunoglobulin; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides and glucose, mannose Or other carbohydrates including dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or non-ionic surfactants Not. Any carrier or excipient is sterile and fluid to the extent that easy syringability exists.
本発明のこの特徴は、医薬的に許容し得る担体又は希釈剤、及び必要に応じて上記に列挙された他の成分中にＭＳＣを組み込むことによって調製される。この例において、ＭＳＣを、ＭＳＣの細胞生存を補助する溶液を含む混合物中に混合してもよい。細胞量は選択される。次に、チャン培地及び所望量のＭＳＣの混合物を選択した溶液へゆっくりと適用することで、ＭＳＣを穏やかに溶液中へ接種する。具体的には、細胞がゲル中に穏やかに混合されるように、自動ピペットから直接溶液上へゆっくりとＭＳＣを放出して、ＭＳＣを接種してもよい。針管腔又は穴が皮下注射の間に剪断力で細胞が損傷を受けないのに十分な直径（例えば、３０ゲージ又はより大きい）である限りは、従来のシリンジを使用し得る。 This feature of the invention is prepared by incorporating MSCs into a pharmaceutically acceptable carrier or diluent and optionally other ingredients listed above. In this example, MSCs may be mixed into a mixture containing a solution that assists MSC cell survival. The amount of cells is selected. The MSC is then gently inoculated into the solution by slowly applying a mixture of Chang's medium and the desired amount of MSC to the selected solution. Specifically, MSCs may be inoculated by slowly releasing MSCs directly from the automatic pipette onto the solution so that the cells are gently mixed into the gel. Conventional syringes can be used as long as the needle lumen or hole is of sufficient diameter (eg, 30 gauge or larger) so that the cells are not damaged by shear forces during subcutaneous injection.
治療
ＭＳＣを含む治療用組成物は、信頼できる医学判断の範囲内で投与され、過度の毒性、炎症、アレルギー反応、又は妥当な利益／危険比に相応の他の合併症なしにヒトの組織と接触させて使用するのに適している。
Therapeutic compositions containing therapeutic MSCs are administered within the scope of reliable medical judgment and are suitable for human tissue without excessive toxicity, inflammation, allergic reactions, or other complications commensurate with a reasonable benefit / risk ratio. Suitable for use in contact.
ＭＳＣは、創傷治癒を改善又は促進するために、治療用組成物中で対象へ投与されてもよい。創傷部位へのＭＳＣの投与によって、細胞は、創傷への体の自然な反応を模倣することとなる。例えば、ＭＳＣは機能して、自然の創傷治癒機構を含む及び／又は補助する他の細胞型、例えば線維芽細胞、炎症細胞又は血管新生のための細胞、へと自然に分化する。 MSCs may be administered to a subject in a therapeutic composition to improve or promote wound healing. By administering MSC to the wound site, the cells will mimic the body's natural response to the wound. For example, MSCs function and naturally differentiate into other cell types that include and / or assist with natural wound healing mechanisms, such as fibroblasts, inflammatory cells or cells for angiogenesis.
投与の前に、物理的又は化学的方法によって創傷を清拭してもよい。 Prior to administration, the wound may be wiped by physical or chemical methods.
一つの態様において、ＭＳＣを接種したマトリックスを創傷へ適用する。 In one embodiment, a matrix inoculated with MSC is applied to the wound.
別の態様において、ＭＳＣをスプレーし又は生体吸収性ゲル若しくは溶液を含む混合物中で創傷へ局所的に適用してもよい。任意に、ＭＳＣの局所的な適用の後にマトリックスを創傷部位に適用し、適切な位置に包帯をしてもよい。 In another embodiment, MSCs may be sprayed or applied topically to the wound in a mixture comprising a bioabsorbable gel or solution. Optionally, after topical application of MSC, the matrix may be applied to the wound site and dressed in place.
更に別の態様において、ＭＳＣを創傷部位で皮下に注射する。 In yet another embodiment, MSCs are injected subcutaneously at the wound site.
本発明の治療用組成物は、ＭＳＣの投薬形態と適合性の方法及び治療的に有効な量において投与される。投与されるＭＳＣの量は、例えば、治療を受ける対象、対象の器官の受容能力、治療用組成物を受け入れる細胞システム又は免疫システム、及び細胞又は組織治療の性質等に依存する。投与に必要な治療用組成物の投薬の量及び頻度は、専門家の判断に依存し、それぞれの個体に固有である。最初の投与の及び後に続く投与のための適切な処方計画もまた様々であるが、マトリックス、ゲル又は注射単独又はいくつかの組み合わせのその後の投与によって望まれる又は示されるような一以上の間隔（例えば、時間、日、週、月又は年）で繰り返される投薬（dose）が後に続く最初の投薬投与を含み得る。 The therapeutic compositions of the invention are administered in a manner compatible with MSC dosage forms and in a therapeutically effective amount. The amount of MSC administered will depend, for example, on the subject to be treated, the capacity of the organ of the subject, the cellular or immune system receiving the therapeutic composition, and the nature of the cellular or tissue treatment. The amount and frequency of therapeutic composition dosage required for administration depends on the judgment of the expert and is specific to each individual. Appropriate regimens for initial administration and subsequent administration also vary, but one or more intervals as desired or indicated by subsequent administration of the matrix, gel or injection alone or some combination ( For example, it may include an initial dose administration followed by a dose repeated in hours, days, weeks, months or years.
一つの態様において、治療用組成物の適用量は、創傷部位毎に約１×１０6細胞〜約１×１０10細胞の範囲でよく、また、投与経路及び創傷部位の大きさに依存してもよい。適用量は創傷部位での投与毎に１×１０10細胞超又は１×１０6細胞未満を含んでもよい。治療は１ヶ月から６ヵ月まで又はそれより長く、毎日、毎週、又は毎月起こり得る適用量の投与を含んでもよい。適用量及び治療は患者の耐性及び治療の進行に一部依存する。 In one embodiment, the amount of therapeutic composition applied may range from about 1 × 10 6 cells to about 1 × 10 10 cells per wound site, depending on the route of administration and the size of the wound site. Also good. The applied dose may comprise more than 1 × 10 10 cells or less than 1 × 10 6 cells per administration at the wound site. Treatment may include administration of an applied dose that may occur daily, weekly, or monthly, from 1 month to 6 months or longer. The dosage and treatment will depend in part on the patient's tolerance and the progress of the treatment.
非制限的な例において、ＭＳＣはマトリックス中に接種された約１×１０6細胞〜約１×１０10細胞の範囲にわたってもよい。接種されたマトリックスは創傷部位に局所的に適用される。適切な適用量は創傷部位において投与毎に１×１０10細胞超又は１×１０6細胞未満を含んでもよい。治療は１ヶ月から６ヵ月まで又はそれより長く、毎日、毎週、又は毎月起こり得る適用量の投与を含んでもよい。一週間後の足場が能力がなければ、その後同数の細胞の局所皮下注射を適用する。適用量は必要に応じて増やしても減らしてもよい。 In a non-limiting example, the MSC may range from about 1 × 10 6 cells to about 1 × 10 10 cells seeded in the matrix. The inoculated matrix is applied topically to the wound site. A suitable application amount may comprise greater than 1 × 10 10 cells or less than 1 × 10 6 cells per administration at the wound site. Treatment may include administration of an applied dose that may occur daily, weekly, or monthly, from 1 month to 6 months or longer. If the scaffold after one week is not capable, then a local subcutaneous injection of the same number of cells is applied. The application amount may be increased or decreased as necessary.
非制限的な例において、ＭＳＣはゲル中に接種された約１×１０6細胞〜約１×１０10細胞の範囲にわたってもよい。適切な適用量は創傷部位において投与毎に１×１０10細胞超又は１×１０6細胞未満を含んでもよい。混合物は創傷部位に局所的に適用される。治療は１ヶ月から６ヵ月まで又はそれより長く、毎日、毎週、又は毎月も適用量の投与を含んでもよい。適用量は必要に応じて増やしても減らしてもよい。 In a non-limiting example, the MSC may range from about 1 × 10 6 cells to about 1 × 10 10 cells seeded in the gel. A suitable application amount may comprise greater than 1 × 10 10 cells or less than 1 × 10 6 cells per administration at the wound site. The mixture is applied topically to the wound site. Treatment may include administration of an applied dose daily, weekly, or monthly for 1 to 6 months or longer. The application amount may be increased or decreased as necessary.
別の非制限的な例において、ＭＳＣは、創傷部位付近又は創傷部位内へ皮下に注射される接種された約１×１０6細胞〜約１×１０10細胞の範囲にわたってもよい。治療は１ヶ月から６ヵ月まで又はそれより長く、毎日、毎週、又は毎月の適用量の投与を含んでもよい。 In another non-limiting example, the MSC may range from about 1 × 10 6 cells to about 1 × 10 10 cells inoculated near the wound site or subcutaneously into the wound site. Treatment may include daily, weekly, or monthly dosage administration from 1 month to 6 months or longer.
別の態様において、適用量投与は、繰り返される投薬間隔におけるマトリックス又はゲルの投与と共に又は代わりの間隔で、後に続くＭＳＣの皮下注射の投与に加えて適用量のＭＳＣを接種されたマトリックス又はゲルを投与することを含んでもよい。 In another embodiment, dosage administration comprises administering a matrix or gel inoculated with a dosage amount of MSC in addition to subsequent administration of subcutaneous injection of MSC with or at alternative intervals of administration of the matrix or gel at repeated dosing intervals. Administration may also be included.
更なる態様において、十分な治療期間、十分な適用量で、接種されたマトリックス、ゲル、及び／又は皮下注射のあらゆる組み合わせにおいて、ＭＳＣを投与してもよい。 In further embodiments, MSCs may be administered in any combination of inoculated matrix, gel, and / or subcutaneous injection at a sufficient treatment period, at a sufficient dose.
マトリックスを用いた投与
この例において、約１×１０6細胞の適用量範囲でＭＳＣを接種された生物分解性マトリックスを、前記形成方法に従って調製し、創傷に適用する又は創傷への投与の前に冷却条件下（２８℃）で保存する。必要に応じて、接種された生物分解性マトリックスの投与の前に創傷を清拭してもよい。接種された生物分解性マトリックスは創傷への局所適用によって投与される。マトリックスは包帯をされ、マトリックスと創傷との接触を保ち、ＭＳＣを創傷部位へ移動させる。
Administration with Matrix In this example, a biodegradable matrix inoculated with MSC at a dosage range of about 1 × 10 6 cells is prepared according to the forming method and applied to the wound or prior to administration to the wound. Store under refrigerated conditions (28 ° C). If necessary, the wound may be wiped prior to administration of the inoculated biodegradable matrix. The inoculated biodegradable matrix is administered by topical application to the wound. The matrix is bandaged, keeping the matrix in contact with the wound and moving the MSC to the wound site.
創傷治癒の進行に依存して６ヵ月まで投与を毎週繰り返す。投与の細胞適用量及び頻度を、患者の耐性及び治療の進行に従って増やしても減らしてもよい。 Dosing is repeated weekly for up to 6 months depending on the progress of wound healing. The cellular dose and frequency of administration may be increased or decreased according to patient tolerance and treatment progress.
発明の製剤に従って、及び創傷状態に依存して、約１×１０6〜約１×１０10ＭＳＣの局所皮下注射によって治療を補ってもよい。皮下注射は、創傷の重大性、治癒の進行及び耐性に依存して毎日、毎週、又は毎月であってよい。 Treatment may be supplemented by topical subcutaneous injection of about 1 × 10 6 to about 1 × 10 10 MSCs according to the formulations of the invention and depending on the wound condition. Subcutaneous injections can be daily, weekly, or monthly depending on the severity of the wound, the progression of healing and tolerance.
ゲルを用いた投与
この例において、約１×１０6細胞の適用量範囲でＭＳＣを接種されたゲルを調製し、創傷に適用する又は投与の前に冷却条件下（２８℃）で保存する。接種されたゲルの投与の前に創傷を清拭してもよい。接種されたゲルは、創傷への噴霧又は直接の適用によって局所的に投与される。ゲルは包帯をされ、ゲル及びＭＳＣと創傷との接触を保ち、創傷部位へＭＳＣを移動させる。
Administration with Gel In this example, a gel inoculated with MSC at a dosage range of about 1 × 10 6 cells is prepared and applied to the wound or stored under cold conditions (28 ° C.) prior to administration. The wound may be wiped prior to administration of the inoculated gel. The inoculated gel is administered topically by spraying or direct application to the wound. The gel is bandaged, keeping the gel and MSC in contact with the wound and moving the MSC to the wound site.
創傷治癒の進行に依存して６ヵ月まで投与を毎週繰り返してもよい。投与の適用量及び頻度を、創傷の重大性、治療の進行、及び耐性に従って増やしても減らしてもよい。 Depending on the progress of wound healing, administration may be repeated weekly for up to 6 months. The dosage and frequency of administration may be increased or decreased according to wound severity, treatment progression, and tolerance.
発明の製剤に従って、及び創傷状態に依存して、約１×１０6〜約１×１０10ＭＳＣの局所皮下注射によって治療を補ってもよい。皮下注射は、創傷の重大性、治癒の進行及び耐性に依存して毎日、毎週、又は毎月であってよい。 Treatment may be supplemented by topical subcutaneous injection of about 1 × 10 6 to about 1 × 10 10 MSCs according to the formulations of the invention and depending on the wound condition. Subcutaneous injections can be daily, weekly, or monthly depending on the severity of the wound, the progression of healing and tolerance.
皮下注射を用いた投与
この例において、約１×１０6細胞の適用量範囲で医薬的に許容し得る賦形剤中に懸濁したＭＳＣを調製し、創傷に適用する又は投与の前に冷却条件下（２８℃）で保存する。皮下注射の前に創傷を清拭してもよい。創傷治癒の進行に依存して６ヵ月まで投与を毎週繰り返してもよい。投与の適用量及び頻度を、創傷の重大性、治療の進行、及び耐性に従って増やしても減らしてもよい。
Administration using subcutaneous injection In this example, MSCs suspended in a pharmaceutically acceptable excipient at a dosage range of about 1 × 10 6 cells are prepared and applied to the wound or cooled prior to administration. Store under conditions (28 ° C). The wound may be wiped before subcutaneous injection. Depending on the progress of wound healing, administration may be repeated weekly for up to 6 months. The dosage and frequency of administration may be increased or decreased according to wound severity, treatment progression, and tolerance.
ＭＳＣを用いるいずれもの治療の間、実験室評価は、ヘマトクリット、ヘモグロビン、平均赤血球容積（ＭＣＶ）、平均赤血球ヘモグロビン（ＭＣＨ）、平均赤血球ヘモグロビン濃度（ＭＣＨＣ）、赤血球分布幅（ＲＤＷ）、血小板数、赤血球数、白血球数（ＷＢＣ）及び差、プロトロンビン時間（ＰＴ）及び国際標準化比（ＩＮＲ）を含む。臨床化学は、カルシウム、ナトリウム、カリウム、塩化物、重炭酸塩、アルブミン、総ビリルビン及び直接ビリルビン、ＡＬＴ、ＡＳＴ、トリグリセリド、コレステロール、血糖、ＢＵＮ、ＡＬＰ、血清クレアチニン、心臓トロポニン、ＣＫ、血清アミラーゼ及び膵リパーゼのようなパラメーターの分析を含む。血清アミラーゼ又はＣＫの上昇があれば、膵アミラーゼ及びＣＫアイソザイム（ＣＫ−ＭＭ、ＣＫ−ＭＢ、ＣＫ−ＢＢ）をそれぞれ分析する。尿検査は、特定の比重、ｐＨ、タンパク質、グルコース、ケトン、潜血、ビリルビン、ウロビリノゲン、及び亜硝酸塩の分析を含む。尿検査に異常があれば、顕微鏡検査（細胞、キャスト（cast）及び結晶の識別）を実施する。 During any treatment with MSC, laboratory evaluations include hematocrit, hemoglobin, mean red blood cell volume (MCV), mean erythrocyte hemoglobin (MCH), mean erythrocyte hemoglobin concentration (MCHC), erythrocyte distribution width (RDW), platelet count, Includes red blood cell count, white blood cell count (WBC) and difference, prothrombin time (PT) and international standardized ratio (INR). Clinical chemistry includes calcium, sodium, potassium, chloride, bicarbonate, albumin, total bilirubin and direct bilirubin, ALT, AST, triglycerides, cholesterol, blood glucose, BUN, ALP, serum creatinine, cardiac troponin, CK, serum amylase and Includes analysis of parameters such as pancreatic lipase. If serum amylase or CK is elevated, pancreatic amylase and CK isozymes (CK-MM, CK-MB, CK-BB) are analyzed, respectively. Urinalysis includes analysis of specific specific gravity, pH, protein, glucose, ketones, occult blood, bilirubin, urobilinogen, and nitrite. If there is an abnormality in the urinalysis, a microscopic examination (cell, cast and crystal identification) is performed.
治療の間、幹細胞産物、サイトカイン、及び成長因子の血漿レベルの測定のために血液試料を回収する。更に、更なるＰＫ分析のために血清試料を回収する（必要であれば）。 During treatment, blood samples are collected for measurement of plasma levels of stem cell products, cytokines, and growth factors. In addition, serum samples are collected (if necessary) for further PK analysis.
いずれの治療に関しても、第一の終点は、重大な不利な事象、投薬を制限する毒性、又はグレード１〜４の検査所見の異常を含む。治療に関する第二の終点は、ベースラインから２８日目までの投薬における変化、治療による影響を受けない患者、治療に対する反応における急速な創傷治癒、急速なＭＳＣ成長（すなわち、治療中の二つの連続した測定における最下点からの１ｌｏｇ増加）、又は治療の１日目から２８日目までの臨床検査における重大な変化のいずれか一つを含む。 For any treatment, the primary endpoint includes a serious adverse event, a dose-limiting toxicity, or an abnormality in grade 1-4 laboratory findings. The second endpoint for treatment was changes in dosing from baseline to day 28, patients unaffected by treatment, rapid wound healing in response to treatment, rapid MSC growth (ie, two successive treatments) 1 log increase from the lowest point in the measured measurement), or a significant change in clinical examination from day 1 to day 28 of treatment.
ラット研究
ラット研究を実施して、マトリックス及びＭＳＣを含む治療用組成物を用いた治療のラットにおける効果を測定した。マトリックスは、４５％酸化再生セルロース及び５５％コラーゲンを有し、殺菌条件下で包装され、凍結乾燥されたＯＲＣプロモグランマトリックスであった。
Rat Study A rat study was performed to determine the effect in rats of treatment with a therapeutic composition comprising a matrix and MSC. The matrix was an ORC promogran matrix with 45% oxidized regenerated cellulose and 55% collagen, packaged under sterilized conditions and lyophilized.
研究のため、４８匹のオスの成体ヌードスプラーグドーリーラット（２５０ｇｍ）を１６匹の３つの実験集団に分けた。前記３グループは、以下のように処置された：（１）普通の創傷をマトリックス（無細胞の皮膚マトリックス、プロモグラン）のみで処置；（２）虚血性創傷をＭＳＣビヒクル（溶液）で処置；及び（３）虚血性創傷をＭＳＣビヒクル及びＭＳＣで処置。痛みのために、それぞれのラットを麻酔し、体重を量ってブプレノルフィン（８〜１２時間間隔で０．０１〜０．０５ｍｇ／ｋｇ2を与える）の皮下注射を投与した。ラットの背中を刈り、１０％ポピドンヨード溶液消毒（ベータダイン）、続いて第三７０％イソプロピルアルコール洗浄と一緒に７０％エタノール洗浄、続いて手術部位へのポビドンヨード溶液の適用の３つの交互の洗浄によって皮膚を消毒した。外科処置の間、３９℃を超えない温度でラットを保った。Chen Cらの創傷修復再生中継器1999;7:486-94において記載されるように、ラットに双茎「皮膚弁」外科処置をした。２．５×１１ｃｍの大きさの皮膚弁用の長方形の型板をラットの背中にトレースした。型板の頭側の基準は肩甲骨の下の角であり、尾側の基準である坐骨結節の面へ広げた。２対の直径６ｍｍのパンチ傷を、型板の基準から３．６ｃｍ及び７．２ｃｍで型板の中線の両側を中心として弁に沿って作成した。バイオプシー（biopsy）を０日試料とし、分析まで−８０℃で冷凍した。
For the study, 48 male nude nude Sprague Dawley rats (250 gm) were divided into three experimental groups of 16 animals. The three groups were treated as follows: (1) normal wound treated with matrix (acellular skin matrix, promogran) only; (2) ischemic wound treated with MSC vehicle (solution); And (3) treating ischemic wounds with MSC vehicle and MSC. Because of the pain, each rat was anesthetized and weighed and administered subcutaneous injections of buprenorphine (giving 0.01-0.05 mg / kg 2 at 8-12 hour intervals). The rat's back is shaved by 10% popidone iodine solution disinfection (betadyne), followed by three alternating washes of 70% ethanol with a third 70% isopropyl alcohol wash, followed by application of povidone iodine solution to the surgical site. The skin was disinfected. Rats were kept at a temperature not exceeding 39 ° C. during the surgical procedure. Rats were treated with a biped “skin flap” surgical procedure as described in Chen C et al. Wound Repair Regenerative Repeater 1999; 7: 486-94. A rectangular template for a flap with a size of 2.5 × 11 cm was traced on the back of the rat. The cranial reference of the template is the lower corner of the scapula and extends to the surface of the sciatic nodule, the caudal reference. Two pairs of 6 mm diameter punch wounds were made along the valve centered on both sides of the midline of the template at 3.6 cm and 7.2 cm from the template reference. Biopsy was taken as
虚血性皮膚弁グループについて、型板の横の線を、皮筋層を貫いて、皮筋層の下にあるフェイシャル（facial）組織の相対的に無血管の面まで切開し、皮膚弁を作成した。骨格筋の上にある緩んだ結合組織の面を通して鈍い切開によって皮膚弁を上げ、次に元の位置に戻して切り込みに沿って９ｍｍのステンレス鋼止め釘で再付着した。 For the ischemic flap group, the lateral line of the template was incised through the cutaneous muscle layer to the relatively avascular surface of the facial tissue beneath the cutaneous muscle layer to create a flap. The flap was raised by a blunt incision through the face of the loose connective tissue overlying the skeletal muscle, then returned to its original position and reattached with a 9 mm stainless steel peg along the incision.
外科処置の日（０日）、その後隔日（２、４、６、８、及び１０日）に、ラットをイソフルレンで麻酔し、体重を量り、創傷包帯を外し、創傷表面領域をフェルトペンを用いて酢酸塩シートでトレースし、試験物質を局所的に創傷へ再適用し、創傷に再び包帯をした。復元後、ラットをケージに戻した。外科処置のときに、創傷をプログマンマトリックスベヒクル及びＭＳＣ又はプログマンマトリックスベヒクル単独で処置し、非接着性包帯（テガダーム、３Ｍ）で包帯をした。ＭＳＣを受け、屠殺を予定していないグループについて、１００μｌにつき１００万ＭＳＣの適用量で、２、４、６、及び８日に創傷へ包帯をするマトリックスへＭＳＣを注入した。それぞれの創傷の酢酸塩トレースを高解像度のＨＰスキャンジェット３Ｃスキャナーを使用してデジタル化した。それぞれの創傷の大きさをＮＩＨイメージＪ（http://rsbweb.nih.gov/ij/）を使用して計算した。平均創傷面積及び平均の標準誤差を計算した。
On the day of surgery (day 0), then every other day (
２、６、８、及び１０日に、創傷の分析のため、それぞれのグループから４匹のラットを屠殺した。安楽死のため、ラットのグループをイソフルレンで麻酔し、その後屠殺した。創傷部位を８ｍｍ穴開け器で切り取り、分析のために組織標本を−８０℃で冷凍した。
On
第一の終点は、隔日の創傷サイズ測定によって評価される創傷治癒の比率であった。 The first endpoint was the rate of wound healing as assessed by alternate day wound size measurements.
０、２、６、８、及び１０日からの創傷の組織像を、パラフィン切片のヘマトキシリン及びエオシン（ＨＥ）染色並びに免疫組織化学染色又は免疫蛍光染色を用いて評価した。免疫組織化学染色を用いて、創傷内のヒト細胞についてヒト主要組織適合抗原を用いて評価した。
Wound histology from
０、２、６、８、及び１０日からの創傷切除物の生化学的分析を、市販のＥＬＩＳＡ分析を用いて、血管内皮成長因子（ＶＥＧＦ）、塩基性線維芽細胞成長因子（ｂＦＧＦ）、及び肝細胞成長因子（ＨＧＦ）について実施した。
Biochemical analysis of wound excisions from
表１に示されるように、創傷面積を、統計的な有意差について、チューキー（Tukey）ＨＳＤ事後検定を用いた一元分散分析を用いて評価した。受傷後の２日目において、無細胞マトリックスビヒクル（プロモグラン）中のＭＳＣで処置された虚血性創傷についての平均創傷面積は、無細胞マトリックスビヒクルのみを用いて処置された虚血性創傷についての又は無細胞マトリックスビヒクルを用いて処置された通常の非虚血性創傷についての平均創傷面積よりも有意に小さかった。ＭＳＣは、虚血性皮膚での創傷治癒において、未処置の創傷での治癒を越える早期のポシティブな効果を提供した。４日目及び６日目において、虚血性創傷における治癒の比率は、通常の創傷及びＭＳＣで治療した虚血性創傷の両方における治癒よりも有意に遅かった。８日目において、３グループ全てにおける創傷は完全な治癒にほぼ達しており、平均創傷面積は有意には違わなかった。１０日目において、全ての創傷が外観上は治癒したように見えた。
As shown in Table 1, wound area was assessed for statistical significance using a one-way analysis of variance using the Tukey HSD post hoc test. On the second day after injury, the mean wound area for ischemic wounds treated with MSCs in a cell-free matrix vehicle (Promogran) is either for ischemic wounds treated with cell-free matrix vehicle alone or It was significantly less than the average wound area for normal non-ischemic wounds treated with a cell-free matrix vehicle. MSC provided an early positive effect on wound healing in ischemic skin over healing in untreated wounds. On days 4 and 6, the rate of healing in ischemic wounds was significantly slower than healing in both normal and MSC-treated ischemic wounds. On
表１：創傷面積の統計分析（有意性（Ｐ＜０．０５））
３つの処置グループについての平均創傷面積と外科処置後の日数のグラフを図２に示す。 A graph of average wound area and days after surgery for the three treatment groups is shown in FIG.
本発明の好ましい態様を示し、記載してきたが、より広い特徴において、本発明から逸脱することなく多くの変更及び修正がなされてもよいことは、当業者にとって明らかであろう。そのため、添付した特許請求の範囲は、発明の真の精神及び範囲内に収まるものとしてそのような変更及び修正の全てに及ぶことを意図されている。 While the preferred embodiment of the present invention has been shown and described, it will be apparent to those skilled in the art that many changes and modifications can be made in broader features without departing from the invention. Accordingly, the appended claims are intended to cover all such changes and modifications as fall within the true spirit and scope of the invention.
Claims (20)
月経由来幹様細胞、及び
治療的に有効な量の月経由来幹様細胞を創傷へ送達して治癒を改善するためのビヒクルであって、治療的に有効な量の月経由来幹様細胞を接種されるビヒクル、
を含むことを特徴とする、前記組成物 A composition for improving wound healing comprising
A vehicle for delivering menstrual stem-like cells and a therapeutically effective amount of menstrual stem-like cells to a wound to improve healing, wherein the therapeutically effective amount of menstrual stem-like cells Vehicle inoculated with cells,
The composition comprising
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
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| US34165210P | 2010-04-01 | 2010-04-01 | |
| US61/341,652 | 2010-04-01 | ||
| PCT/US2011/030939 WO2011123779A1 (en) | 2010-04-01 | 2011-04-01 | Cell therapy to improve wound healing and related compositions and methods |
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| JP2013523774A true JP2013523774A (en) | 2013-06-17 |
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| JP2013502888A Withdrawn JP2013523774A (en) | 2010-04-01 | 2011-04-01 | Cell therapy to improve wound healing and related compositions and methods |
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| EP (1) | EP2553089A4 (en) |
| JP (1) | JP2013523774A (en) |
| CA (1) | CA2795248A1 (en) |
| WO (1) | WO2011123779A1 (en) |
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| WO2016036322A1 (en) | 2014-09-04 | 2016-03-10 | Kemijski inštitut | Cell-based device for local treatment with therapeutic protein |
| CN106176813A (en) * | 2016-07-28 | 2016-12-07 | 广州赛莱拉干细胞科技股份有限公司 | A kind of compositions repairing skin ulcer and preparation method thereof |
| CN106074605A (en) * | 2016-07-28 | 2016-11-09 | 广州赛莱拉干细胞科技股份有限公司 | A kind of compositions repairing skin ulcer and preparation method thereof |
| CN110693911A (en) * | 2019-10-30 | 2020-01-17 | 南通大学 | Menstrual blood-derived endometrial stem cell preparation and preparation method and application thereof |
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| JP2006230316A (en) * | 2005-02-25 | 2006-09-07 | Japan Health Science Foundation | Cell having wound healing ability without scar and method for preparing the same |
| US20080241113A1 (en) * | 2007-03-01 | 2008-10-02 | Cryo-Cell International, Inc. | Procurement, isolation and cryopreservation of endometrial/menstrual cells |
| US20080279833A1 (en) * | 2007-05-10 | 2008-11-13 | Matheny Robert G | Laminate sheet articles for tissue regeneration |
| WO2008148105A1 (en) * | 2007-05-25 | 2008-12-04 | Medistem Laboratories, Inc. | Endometrial stem cells and methods of making and using same |
| US8084057B2 (en) * | 2007-07-17 | 2011-12-27 | University Of Massachusetts | Compositions and methods for wound healing |
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- 2011-04-01 WO PCT/US2011/030939 patent/WO2011123779A1/en active Application Filing
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| WO2011123779A1 (en) | 2011-10-06 |
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| CA2795248A1 (en) | 2011-10-06 |
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