JP2019010089A - 人工皮膚製造方法および人工皮膚 - Google Patents
人工皮膚製造方法および人工皮膚 Download PDFInfo
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Abstract
Description
真皮(dermis)は、連結組織からなる表皮下の皮膚層で、緩衝作用をして身体を圧力と張力(stress and strain)から保護する。真皮は、基底膜(basement membrane)を通じて表皮と固く連結されている。真皮は、その構造に内在している血管、神経などの多様な付属物を支持する基質を供給する。
実施例1
(1)1段階:真皮模写層の製造
線維芽細胞とコラーゲンを混合して真皮模写層を製作した後、1週間培養した。
前記1段階以降、前記真皮模写層上に角化細胞(HEKn、Thermo Fisher)を載せて2日間培養した。
前記2段階以降、前記真皮模写体の下の部分に第1培地(CnT−3D−PR、CellnTEC)が接触するように設け、表皮層部分は空気に露出させる空気露出8日間培養を行った。
前記3段階以降、第2培地で、追加で空気露出6日間培養を行って人工皮膚モデルを得た。第2培地の組成は、DMEM培地(Welgene、LM001−05)とF12培地(Welgene、LM010−01)を2.5:1比率で混合し、ここにヒドロコルチゾン(hydrocortisone)、トリヨードサイロニン(Triiodothyronine)、コレラ毒素(Cholera toxin)、アスコルビン酸(ascorbic acid)および塩化カルシウム(CaCl2)を含むものである。
得られた人工皮膚モデルの顕微鏡断面写真を図2に示す。
第2培地での培養段階(4段階)を経ないことを除いては、実施例1と同様にして人工皮膚を製作した。
得られた人工皮膚モデルの顕微鏡断面写真を図3に示す。
実施例1および比較例1で得られた人工皮膚モデルの断面を顕微鏡で観察する。
Claims (13)
- 線維芽細胞およびコラーゲンを混合して真皮模写層を形成する段階、
前記真皮模写層上に角化細胞を適用した後に培養する段階、
前記真皮模写層を角化細胞培地である第1培地で培養する段階、そして
前記第1培地で培養された真皮模写層をDMEM培地およびF12培地が混合された第2培地で培養する段階
を含む
人工皮膚製造方法。 - 前記角化細胞は、メラニン形成細胞の幹細胞を含む、請求項1に記載の人工皮膚製造方法。
- 前記第2培地は、前記メラニン形成細胞の幹細胞の分化を誘導する成分を含有する、請求項2に記載の工皮膚製造方法。
- 前記第1培地は、前記真皮模写層の下面に接触するように設けられ、当該真皮模写層において前記第1培地が設けられた面の反対面は空気に露出させる、請求項1に記載の人工皮膚製造方法。
- 前記第2培地での培養段階は、前記第1培地での培養段階が終結した後直ちに続いて行われる、請求項1に記載の人工皮膚製造方法。
- 前記第1培地での培養期間および前記第2培地での培養期間の合算は、20日以内である、請求項1に記載の人工皮膚製造方法。
- 前記第1培地での培養期間は、前記第2培地での培養期間と比較して長いかまたは同じである、請求項6に記載の人工皮膚製造方法。
- 前記第2培地は、前記DMEM培地および前記F12培地が1:1乃至4:1の比率で混合されたものである、請求項1に記載の人工皮膚製造方法。
- 前記第2培地は、ヒドロコルチゾン(hydrocortisone)、トリヨードサイロニン(Triiodothyronine)、コレラ毒素(Cholera toxin)、アスコルビン酸(ascorbic acid)および塩化カルシウム(CaCl2)の中から選択される一つまたは二つ以上をさらに含む、請求項1に記載の人工皮膚製造方法。
- 前記第2培地での培養段階は、前記第1培地で培養された真皮模写層が空気に露出された状態で行われる、請求項1に記載の人工皮膚製造方法。
- 真皮模写層および前記真皮模写層上に位置する表皮模写層を含む人工皮膚であって、
前記表皮模写層は、前記真皮模写層に隣接している表皮基底層部分および空気中に露出されている角質層部分を含み、
前記角質層部分には角化細胞が存在すると共に、前記表皮基底層部分にはメラニン形成細胞が存在し、これら角質層部分に存在する角化細胞と前記表皮基底層部分に存在するメラニン形成細胞とは、同一ヒト由来のものである
人工皮膚。 - 色素化調整剤の効能評価方法であって、
請求項1乃至10のいずれか一項に記載の方法により製造された人工皮膚、または請求項11に記載の人工皮膚に、前記色素化調整剤を適用する段階、そして
前記色素化調整剤が適用された人工皮膚の色素沈着程度と前記色素化調整剤が適用されていない人工皮膚の色素沈着程度とを比較する段階
を含む
色素化調整剤の効能評価方法。 - 前記色素化調整剤は、前記人工皮膚と直接接触により適用される、請求項12に記載の色素化調整剤の効能評価方法。
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