Title
画像濃度を用いた細胞凝集塊形態特性の評価の検討
Author(s)
渡部 俊樹, 下戸 健
Citation
福岡工業大学総合研究機構研究所所報 第2巻 P95-P98
Issue Date
2020-2
URI
http://hdl.handle.net/11478/1489
Right
Type
Departmental Bulletin Paper
Textversion
Publisher
福岡工業大学 機関リポジトリ
FITREPO
⏬ീ⃰ᗘࢆ⏝࠸ࡓ⣽⬊จ㞟ሢᙧែ≉ᛶࡢホ౯ࡢ᳨ウ
Ώ㒊 ಇᶞ㸦Ꮫ㝔ᕤᏛ◊✲⛉ሗࢩࢫࢸ࣒ᕤᏛᑓᨷ㸧
ୗᡞ 㸦ሗᕤᏛ㒊ሗࢩࢫࢸ࣒ᕤᏛ⛉㸧
Evaluation of Spheroid Morphological Characteristics 8sing the Image Density
Toshiki WATANABE 㸦Information and Systems Engineering, Graduate School of Engineering㸧 Takeshi SHIMOTO 㸦Department of Information and Systems Engineering, Faculty of Information Engineering㸧
Abstract
Our research group has established a technique to make three-dimensional cell constructs using only cells. The cell constructs are made using spheroids consisting of only cells. The quality of the spheroids is important as it is necessary to provide reproducible spheroids. Therefore, it is necessary to morphologically evaluate spheroids non-invasively. The quality of spheroids was evaluated regarding area, diameter, and circularity. However, the concentration distribution of the spheroids had not previously been visualized. This study aimed to visualize the cell distribution inside spheroids using pseudo-color display processing. By applying pseudo-color processing to the grayscale spheroid images, we succeeded in visually grasping cell distribution and shape. In the future, we will actually observe inside the spheroid and aim to further improve the accuracy by correlating with the specific conditions.
Keywords㸸Tissue engineering, Spheroid morphological characteristics, Pseudo-color image processing, Spheroid morphology evaluation system
1. ⥴ゝ ⏕་⒪ࡣ㸪ᝈࡼࡾᶵ⬟࡞ࡗࡓ⮚ჾࡸ⤌⧊ ࡢᶵ⬟ᅇࢆ⾜࠺⒪ἲ࡛࠶ࡾ㸪ᚑ᮶ࡢ⒪࡛ࡣᅔ㞴࡞㑇 ఏᛶᝈࡢ⒪ἲࡋ࡚ᮇᚅࡉࢀ࡚࠸ࡿ㸬ᶵ⬟ࡋ࡞ࡃ࡞ࡗ ࡓ⮚ჾ࠾ࡼࡧ⤌⧊ࡢ୍㒊ࢆᕤᏛⓗ࡞ࣉ࣮ࣟࢳࡼࡗ࡚ಟ ࣭⨨ࡍࡿTissue Engineering ࡀ 1993 ᖺ⡿ᅜ࡛ᥦၐ ࡉࢀ1)㸪⣽⬊࣭ᡂ㛗ᅉᏊ࣭Scaffold ࡢ 3 ࡘࡢ⤌ࡳྜࢃࡏࡼ ࡾయእ࡛ேᕤⓗ⣽⬊ᵓ㐀యࢆᙧᡂࡍࡿ◊✲ࡀ⾜ࢃࢀ࡚࠸ ࡿ㸬ࡇࢀࡲ࡛❧యᵓ㐀యࢆస〇ࡍࡿ᪉ἲࡋ࡚㸪௵ពࡢ ⨨⣽⬊ࢆ✀ࡋ࡞ࡀࡽ✚ᒙࡉࡏࡿࣂ࢜ࣉࣜࣥࢸ ࣥࢢᢏ⾡ࢆ⏝࠸ࡓ᪉ἲ2-5)㸪⣽⬊ࢆࣇࣂ࣮≧ᙧᡂࡋ࡚ ⧊ࡗࡓࡾ᮰ࡡࡓࡾࡍࡿࡇ࡛స〇ࡍࡿ᪉ἲ6)㸪⣽⬊ࢆ୍య ࡉࡏࡓ⣽⬊ࣅ࣮ࢬࢆ⏝࠸࡚స〇ࡍࡿ᪉ἲ࡞ࡀሗ࿌ࡉࢀ࡚ ࠸ࡿ㸬3 ḟඖᵓ㐀యࡢᐇ⏝㛵ࡋ࡚ࡣ㸪ࢥ࣮ࣛࢤࣥࢤࣝෆᇵ 㣴ࡋࡓ㌾㦵ᵝ⛣᳜ 7)ࢆࡣࡌࡵࡋࡓ㌾㦵⏕ᢏ⾡ࡢᐇ⏝ 8)ࡀᩓぢࡉࢀࡿࡀ㸪ึᮇⓗ࡞㌾㦵ᦆയࡢࡳࡋ㐺⏝࡛ࡁ࡚ ࠸࡞࠸㸬ᮏ◊✲ࢢ࣮ࣝࣉ࡛ࡣ㸪㞄᥋ࡍࡿ⣽⬊จ㞟ሢྠኈࡣ ⼥ྜࡍࡿ࠸࠺≉ᛶ㸦ᅗ1㸧ࢆ⏝ࡋ࡚㸪⣽⬊จ㞟ሢࢆ௵ព ࡢ 3 ḟඖ✵㛫㓄⨨ࡍࡿ⊂⮬ࡢᢏ⾡࡛⣽⬊ࡢࡳࢆ⏝࠸ࡓ❧ యᵓ㐀యࡢస〇ἲࢆ☜❧ࡋ࡚࠸ࡿ㸬㔞ࡢ⣽⬊จ㞟ሢࢆࣈ ࣟࢵࢡࡢࡼ࠺✚ࡳ㔜ࡡ࡚❧యᵓ㐀యࢆస〇ࡍࡿࡓࡵ㸪ྠ ୍ᙧ≧ࡢ⣽⬊จ㞟ሢࢆᚓ࡞ࡅࢀࡤ࡞ࡽ࡞࠸㸬ࡉࡽ㸪⣽⬊ จ㞟ሢෆ㒊ࡢほᐹࡢࡓࡵ◚ቯࡋ࡚ࡋࡲ࠺㸪❧యᵓ㐀య ࡢస〇⏝࡛ࡁ࡞ࡃ࡞ࡿࡓࡵ㸪⤒ⓗࡘ㠀く࡛ࡢ⣽ ⬊จ㞟ሢࡢホ౯ࡀồࡵࡽࢀ࡚࠸ࡿ㸬 ᮏ◊✲ࢢ࣮ࣝࣉ࡛ࡣ㸪࣮࢜ࢺ࣓࣮ࢩࣙࣥࡼࡿ㧗ရ㉁࡞ ⣽⬊จ㞟ሢࡢస〇࠾ࡼࡧᥦ౪ࢆ┠ⓗ◊✲ࢆ⾜ࡗ࡚࠸ࡿ㸬 ࡇࢀ㛵ࡋ㸪⣽⬊จ㞟ሢࡢ㠀くⓗ࡞ホ౯↔Ⅼࢆᙜ࡚㸪 ⣽⬊จ㞟ሢᙧែホ౯ࢩࢫࢸ࣒⣽⬊จ㞟ሢゎᯒࢯࣇࢺ࢙࢘ ࢆ㛤Ⓨࡋࡓ㸬96 ࢙࢘ࣝࣉ࣮ࣞࢺࢆ⮬ື࡛ᙳࢆ⾜࠺ࡇ ࢆྍ⬟ࡋ㸪⣽⬊จ㞟ሢࡢ㠃✚㸪┤ᚄ࠾ࡼࡧᙧᗘࢆ⟬ฟ ࡋရ㉁ࢆホ౯ࡍࡿࡇࡀ࡛ࡁࡿ㸬ࡋࡓࡀࡗ࡚㸪⣽⬊จ㞟ሢ ࡀእẼゐࢀࡿ㛫ࢆ▷⦰ࡋࢲ࣓࣮ࢪࢆ㍍ῶ࡛ࡁࡿࡇࡔ ࡅ࡛ࡣ࡞ࡃ㸪࣮࢜࣌ࣞࢱࡢ㈇ᢸ㍍ῶసᴗࡢຠ⋡⧅ࡆ ࡿࡇࡀ࡛ࡁࡓ㸬ࡋࡋ࡞ࡀࡽ㸪⏬ീ⃰ᗘࢆ⏝࠸࡚⣽⬊จ 㞟ሢࡢᙧ≧ࢆᇶ⣽⬊ศᕸࢆどぬࡋࡓࡇࡣࡇࢀࡲ࡛ ࡞ࡗࡓ㸬 Volocity㸦Perkin Elmer ♫㸧࡞ࡢ 3 ḟඖ⏬ീᵓ⠏㸪ゎᯒࢯ ࣇࢺ࢙࢘ࢆ⏝࠸ࡿࡇࡼࡾ㸪❧య⏬ീࡋ࡚ࢥࣥࣆࣗ ࣮ࢱୖ♧ࡍࡇࡀ࡛ࡁࡿᢏ⾡ࡀሗ࿌ࡉࢀ࡚࠸ࡿࡀ㸪ࢥࢫ ࢺࡀࡿ࠸࠺ၥ㢟Ⅼࡀ࠶ࡿ㸬ࡑࡇ࡛ᮏ◊✲࡛ࡣ㸪ᙳ ࡋࡓ⣽⬊จ㞟ሢ⏬ീᑐࡋ࡚⏬ീฎ⌮ࢆࡋ㸪⣽⬊ศᕸࢆ どぬⓗᢕᥱࡍࡿࡇࢆ᳨ウࡋࡓ㸬
Flg.1 Spheroids created in 96-well plates
Spheroid 96well plate
Ώ㒊 ಇᶞ㸪ୗᡞ 2 ᑐ㇟࠾ࡼࡧ᪉ἲ ࠑ2.1ࠒ ⣽⬊จ㞟ሢᙧែホ౯ࢩࢫࢸ࣒ 㛤Ⓨࡋࡓ⣽⬊จ㞟ሢᙧែホ౯ࢩࢫࢸ࣒ࡢᅗ2 ♧ࡍ㸬స 〇ࡋࡓ⣽⬊จ㞟ሢࢆᙳࡍࡿࡓࡵࡢࢩࢫࢸ࣒㸪ᙳࡋࡓ ⏬ീࢆฎ⌮ࡍࡿࢩࢫࢸ࣒ศࢀ࡚࠸ࡿ㸬⣽⬊จ㞟ሢࡣ96 ࢙࢘ࣝࣉ࣮ࣞࢺ㸦SUMILON PrimeSurface®, Sumitomo Bakelite㸧୍ᐃᩘࡢ⣽⬊ࢆ✀ࡋ࡚స〇ࡉࢀࡿ㸬1 ࡘࡢ࢘ ࢙ࣝ 1 ࡘࡢ⣽⬊จ㞟ሢࡀస〇ࡉࢀࡿ㸬ᆺࡢ⣽⬊ᵓ㐀య ࡢస〇࡞ࡿ㸪㔞ࡢ96 ࢙࢘ࣝࣉ࣮ࣞࢺࡀᚲせ࡞ࡾ㸪 ࡚ࡢࣉ࣮ࣞࢺᑐࡋ࡚㢧ᚤ㙾࡛☜ㄆࡋ࡞ࡀࡽᡭ᥈ࡾ࡛⣽ ⬊จ㞟ሢࢆ᥈ࡋฟࡋ࡚ᙳࡋ㸪ᚓࡽࢀࡓ⏬ീࢆゎᯒࡍࡿࡢ ࡣ㸪సᴗ⪅⭾࡞㈇ᢸࡀࡿ㸬ࡑࡢࡓࡵ㸪ᙳゎᯒ ࢆ⮬ືࡉࡏࡓ㸬2 ྎࡢࢫࣛࢲ㸦EZSM3-D015-ARAK, EZSM4- D010-ARAK, ORIENTALMOTOR ♫〇㸧ࢆ┤ ⤌ࡳྜࢃࡏࡓࡶࡢ㸪96 ࢙࢘ࣝࣉ࣮ࣞࢺᑐࡋ࡚ᆶ┤᪉ ྥ࣓࢝ࣛࣛࢺࢆྲྀࡾࡅ㸪࣓࢝ࣛࢆX ㍈ Y ㍈᪉ྥ ࡑࢀࡒࢀࡢ࢙࢘ࣝࡲ࡛⨨ྜࢃࡏ࡛ࡁࡿࡼ࠺ࡋࡓ㸬2 ྎࡢ ࢫࣛࢲࡣศゎ⬟20ȣm㸪㉮ࡾᖹ⾜ᗘ 30ȣm㸪⧞ࡾ㏉ࡋ ⨨Ỵࡵไᗘs20ȣm ࡛࠶ࡾ㸪ྛ࢙࢘ࣝࡢ⣽⬊จ㞟ሢయࡀ ᙳ࡛ࡁࡿࡼ࠺㸪ᚤㄪᩚࡍࡿࡇࡀ࡛ࡁࡿ㸬 XY ࢫࣛ ࢲࡀไᚚࡍࡿ㠃ᑐࡋ࡚ἲ⥺᪉ྥZ ࢫࢸ࣮ࢪ㸦ZSGB60, MISUMI ♫〇㸧ࢆタࡅ㸪࣓࢝ࣛࢆྲྀࡅࡿࡇࡼࡾ㸪ᡭ ື࡛ᙳࣆࣥࢺࢆྜࢃࡏࡽࢀࡿࡼ࠺ࡋࡓ㸬⣽⬊ࢆ ᙳ ࡍࡿ࢝ ࣓ࣛࡣ USB ࣓࢝ࣛ 3.0㸦EMYC-CB500M3㸪 MIAUMI ♫〇㸧ࢆ⏝ࡋࡓ㸬ᫎീฟຊ᭷ຠ⏬⣲ᩘࡣ 2592 1944pixel 㸪 ⏬ ⣲ ࢧ ࢬ ࡣ 2.2 2.2um 㸪 ឤ ග ᗘ ࡣ 1.4V/lux-sec ࡛࠶ࡿ㸬⏬ീྲྀᚓ࡛ࡣ㸪96 ࢙࢘ࣝࣉ࣮ࣞࢺ ࠾ࡅࡿྛ⣽⬊จ㞟ሢࡢࢻࣞࢫࡢྲྀᚓ⣽⬊จ㞟ሢᙳ ࢆྠ⾜࠼ࡿࡼ࠺࡞ࢯࣇࢺ࢙࢘ࡋࡓ㸬㢧ᚤ㙾➼࡛ࡢ ほᐹ࡛ࡣ㸪࣓࢝ࣛࡀᅛᐃࡉࢀ࡚࠸ࡿࡓࡵ㸪ࣉ࣮ࣞࢺࢆື ࡋ࡚ほᐹࡍࡿ㸬ࡑࡢሙྜ㸪ᇵ㣴ᾮࡸ⣽⬊จ㞟ሢࡀືࡃࡓࡵ㸪 Ᏻᐃࡍࡿࡲ࡛ᚅࡘᚲせࡀ࠶ࡿ㸬ࡇࢀᑐࡋ㸪ᮏࢩࢫࢸ࣒ࡣ ࣓࢝ࣛࢆືࡍࡼ࠺ࡋ࡚࠾ࡾ㸪⣽⬊จ㞟ሢእຊࢆ࠼ ࡎほᐹ࠾ࡼࡧゎᯒࡍࡿࡇࡀ࡛ࡁࡿ㸬 ⣽⬊จ㞟ሢᙧែホ౯ࢩࢫࢸ࣒࡛ᙳࡋࡓ⣽⬊จ㞟ሢࡢ⏬ ീᑐࡋ࡚ᐃ㔞ホ౯ࢆ⾜࠺ࡓࡵ㸪⏬ീฎ⌮ࡼࡾ⣽⬊จ 㞟ሢࡢ⃰ᗘศᕸࢆどぬࡍࡿࡇࢆヨࡳࡓ㸬⣽⬊จ㞟ሢࡣ ⣽⬊ࡀจ㞟ࡋ࡚ࡃࡓࡵ⌫≧࡞ࡿ㸬ࡑࡢࡓࡵ㸪ᙧ≧ࢆほᐹ ࡍࡿࡓࡵࡣ᭱ప2 ᪉ྥࡽࡢᙳࡀᚲせ࡛࠶ࡿ㸬ࡋࡋ㸪 96 ࢙࢘ࣝࣉ࣮ࣞࢺࢆᶓࡽᙳࡍࡿሙྜ㸪ྛ࢙࢘ࣝࡣᗏࡀ ⌫≧࡞ࡗ࡚࠸ࡿࡇྛ࢙࢘ࣝࡢ⣽⬊จ㞟ሢࣆࣥࢺࢆ ྜࢃࡏࡽࢀ࡞࠸ࡇࡽ㸪ᗏ᪉ྥࡽࡢ1 ᪉ྥᙳࡋࡓ㸬 ⣽⬊จ㞟ሢࡣ⌮ㄽⓗࡣ⌫≧࡞ࡿࡇࢆ⪃៖ࡋࡓࢩ࣮ࣗ ࢻ࣮࢝ࣛฎ⌮ࢆࡋࡓ㸬⣽⬊จ㞟ሢࡣእ࿘㒊ࡢ⣽⬊ࡀ୰ᚰ 㒊ᕳࡁ㎸ࡲࢀࡿࡇࡼࡗ࡚จ㞟ࡍࡿࡓࡵ㸪୰ᚰ㒊ࡢ⃰ ᗘࡀ᭱ࡶ㧗ࡃ㸪እ࿘㒊ࡣ⃰ᗘࡀపࡃ࡞ࡿ⪃࠼ࡽࢀࡿ㸬ண ഛᐇ㦂ࡽࢫࣇ࢙ࣟࢻ୰ᚰ㒊ศࡢᇶ‽್ࢆ30㸪እ࿘㒊ศ ࡢᇶ‽್ࢆ176 ࡋࡓ㸬⃰ᗘࡢᇶ‽ࡣ⫼ᬒࡢ⃰ᗘ್ࡋࡓ ࡀ㸪⏬ീࡼࡗ࡚⃰ᗘ್㐪࠸ࡀ☜ㄆ࡛ࡁࡓࡓࡵ㸪እ࿘㒊 ࡢᇶ‽್ࢆ⏬ീẖኚືࡉࡏࡿタᐃࡋࡓ㸬⏬ീᅄ㝮10 10 pixel ࡢ㡿ᇦࡢ 100 ಶࡎࡘィ 400 ಶࡢ㜈್ࡢᖹᆒ್ࢆ⟬ ฟࡋ㸪እ࿘㒊ศࡢᇶ‽್Xࡋࡓ㸬ࢩ࣮ࣗࢻ࣮࢝ࣛฎ⌮ࡣ㸪 ⫼ᬒᖹᆒ㜈್ࢆ⏝࠸࡚10 ศࡉࢀࡓ㜈್ࢆ㸪ࡢ᪉⛬ᘧ ࡼࡾ⟬ฟࡋࡓ㜈್bnᇶ࡙࠸࡚Ⰽࡅࢆ⾜ࡗࡓ㸦ᅗ3㸧㸬 ᙳࡋࡓ⣽⬊จ㞟ሢ⏬ീ㛤Ⓨࡋࡓࢯࣇࢺ࢙࡛࢘ࢩ࣮ࣗࢻ ࣮࢝ࣛࢆ㐺⏝ࡉࡏ㸪⣽⬊จ㞟ሢࡢ⃰ᗘศᕸኚࢆ⤒ⓗ ほᐹࡋࡓ㸬
Fig.3 Pseudo-color based on the threshold calculated from equation of circle
Light Dark
Gray scale chart
Pseudo-color chart 30 X Threshold ؟ ࠑ2.2ࠒ ⣽⬊จ㞟ሢࡢᙧែホ౯ ⣽⬊ࡣṇᖖ࢘ࢧࢠ㛫ⴥ⣔ᖿ⣽⬊ࢆ⏝࠸ࡓ㸬⥅௦ࡣ࢝ࣝࢳ ࣮ࣕࢹࢵࢩࣗ㸦ȍ150mm, H25mm, CORNING㸧ࢆ⏝࠸
Fig.2 The spheroid morphology evaluation system M onitor Personal Computer I/O board USB Camera Software with C# -Hardware control -Image processing -Analysis Driver Y-Slider 2-axis Controller Driver X-Slider Z-Slider (Manual) Light source device Spheroids
96-well plate is fixed Shot images
Light source device 96-well plate
XY-Slider Camera
ࡓ㸬ᇵ㣴ᾮࡣDMEM㸦Dulbecco's Modified Eagle Medium, gibco㸧 Penicillin-Streptomycin Solution ࢆ 1%㸪Fetal bovine serum ࢆ 10%ῧຍࡋࡓࡶࡢࢆ⏝ࡋࡓ㸬⣽⬊ᇵ㣴ࡣ ᑓ㛛ࡢᢏ⾡ᢏ⬟ဨࡀ⾜ࡗࡓ㸬ᮏ◊✲࠾ࡅࡿ⣽⬊ᇵ㣴ᢏ⾡ ⪅ࡣ㸪Ꮫ➼ࡢ᭷㈨᱁⪅࡛ࡣ࡞࠸ࡀ㸪⣽⬊ᇵ㣴ࡢᇶᮏࢆ⌮ ゎࡋ㸪⣽⬊ᰴࢆ㐺ษ࡞᪉ἲ࡛ᇵ㣴࡛ࡁࡿࡶ㸪24 ࣨ᭶ ௨ୖࡢ⣽⬊ᇵ㣴ࡢ⤒㦂ࢆ᭷ࡋ࡚࠸ࡿ⪅ࡋࡓ㸬⣽⬊ࡣࢹ ࢵࢩࣗෆ࡛ࢥࣥࣇ࢚ࣝࣥࢺࡍࡿ᥋ゐ㜼ᐖࢆ㉳ࡇࡋቑṪࢆ Ṇࡵ࡚ࡋࡲ࠺ࡓࡵ㸪ࢥࣥࣇ࢚ࣝࣥࢺࡍࡿ๓⥅௦ࢆ⾜࠸࡞ ࡀࡽᇵ㣴ࢆ㐍ࡵࡓ㸬ᇵᆅࡢࡣ2 ᪥ 1 ᅇ⾜࠸㸪ࢫࣇ࢙ ࣟࢻᇵ㣴ᚲせ࡞⣽⬊ᩘ㐩ࡍࡿࡲ࡛ᇵ㣴ࢆ⾜ࡗࡓ㸬⣽ ⬊ᵓ㐀యࡣᮏ◊✲ࢢ࣮ࣝࣉ࡛㛤Ⓨࡋ࡚࠸ࡿ⮬ື⣽⬊จ㞟ሢ ᇵ㣴ࢩࢫࢸ࣒ࢆ⏝࠸ࡓ㸦ᅗ 4㸧㸬⣽⬊ᩘࡣᮏ◊✲ࡢ⣽⬊ᵓ㐀 యస〇࠾࠸࡚⏝㢖ᗘࡀ㧗࠸ 2104㸪3104㸪4104 cell/well ࡋ㸪⥅௦ᩘࡣ 7 ࡋࡓ㸬⣽⬊จ㞟ሢࢆᙳࡍࡿ ᮇ㛫ࡣ5 ᪥㛫ࡋࡓ㸦⾲ 1㸧㸬 Medium Cell suspension for Spheroids
Scalar robot 2¼2 channel pipette
Cell suspension
Fig. 4 The automatic spheroids culture system Input the cell count of the cell suspension which prepared
Change of medium in 96-well plate
Dispense the prepared cell suspension into a 96-well plate The cell suspension was arbitrarily
adjusted with cells and medium
Table 1 Spheroids manufacturing conditions
Cell Rabbit mechanical stem cell, (rMSC) Operations Automatic spheroids culture system Passage number P7
Cell number (cell/well) 2¼104, 3¼104, 4¼104
Photography period 5 days
3. ⤖ᯝ࠾ࡼࡧ⪃ᐹ ⣽⬊จ㞟ሢᙧែホ౯ࢩࢫࢸ࣒࡛ᙳࡋࡓ⣽⬊จ㞟ሢࡢ⏬ ീࢆᅗ5 ♧ࡍ㸬㛫⤒㐣కࡗ࡚㸪จ㞟ࡋ࡚࠸ࡿᵝᏊࡣ ほᐹࡍࡿࡇࡀ࡛ࡁࡿࡀ㸪⃰ᗘࡢኚࡘ࠸࡚ࡣ㸪⫗║ⓗ ࡣุ᩿ࡍࡿࡇࡀ࡛ࡁ࡞ࡗࡓ㸬 ᅗ 5 ࡢ⏬ീᑐࡋ㸪㛤Ⓨࡋࡓࢯࣇࢺ࢙࢘ࢆ⏝࠸࡚ࢩࣗ ࣮ࢻ࣮࢝ࣛฎ⌮ࢆࡋࡓ⏬ീࢆᅗ6 ♧ࡍ㸬స〇ࡋ࡚ 1 ᪥ ┠2 ᪥┠ࡢ 2104㸪3104 cell/well ࡢ⣽⬊จ㞟ሢࡣ㸪୰ ᚰ㒊ࡽእ࿘㒊ࡅ࡚⥳ྠࡢ㡿ᇦࡀᗈࡗࡓ㸬ࡇࢀࡣ㸪 ⣽⬊จ㞟ሢࡀࡲࡔ⌫≧㞟ࡲࡗ࡚࠸࡞࠸⪃࠼ࡽࢀࡿ㸬ࡇ ࢀᑐࡋ4104 cell/well ࡢ⣽⬊จ㞟ሢ࡛ࡣ㸪⣽⬊ᩘࡀከ࠸ ࠸࠺ࡇࡶ࠶ࡿࡀ㸪㯤Ⰽࡢ㡿ᇦࡶ࠶ࡾ㸪⌫≧ᙧᡂࡋࡣ ࡌࡵ࡚࠸ࡿࡇࡀ⪃࠼ࡽࢀࡿ㸬ࡉࡽ㸪⣽⬊ᩘࡀከ࠸᪉ࡀ άⓎจ㞟ࡋ࡚࠸ࡿࡇࡶ⪃࠼ࡽࢀࡿ㸬స〇ࡋ࡚ 3 ᪥┠ࡢ ⣽⬊จ㞟ሢࡣ㠃✚ࡀᑠࡉࡃ㸪㯤Ⰽࡢ㡿ᇦࡀᗈࡃ࡞ࡾ㸪ᶳⰍ ࡢ㡿ᇦࡶ☜ㄆ࡛ࡁࡓ㸬ࡇࢀࡣ⣽⬊ࡀจ㞟ࡋ㸪⣽⬊ࡢᐦᗘࡀ 㧗ࡃ࡞ࡿྠ⌫≧࡞ࡗ࡚࠸ࡿ⪃࠼ࡽࢀࡿ㸬స〇ࡋ ࡚4 ᪥┠ 5 ᪥┠ࡢ⣽⬊จ㞟ሢࡣ㸪㯤Ⰽࡢ㡿ᇦࡀ⊃ࡃ࡞ࡾ㸪 ⥳Ⰽࡢ㡿ᇦࡀᗈࡀࡗ࡚࠸ࡿࡇࡀ☜ㄆࡉࢀࡓ㸬ࡇࢀࡣ㸪⣽ ⬊จ㞟ሢෆࡢ⣽⬊ࡀṚ⁛ࡍࡿࢿࢡ࣮ࣟࢩࢫ⌧㇟ࡀ㉳ࡁ࡚࠸ ࡿྍ⬟ᛶࡀ⪃࠼ࡽࢀࡿ㸬ࢿࢡ࣮ࣟࢩࢫ⌧㇟ࡀ㉳ࡁࡿ㸪Ṛ ⁛⣽⬊ࡀᾮ≧࡞ࡾእ㒊ฟࡉࢀࡿ㸬ࡋࡓࡀࡗ࡚㸪ෆ 㒊࠶ࡗࡓ⣽⬊จ㞟ሢ㒊ศࡀ✵Ὕ࡞ࡾ㸪⣽⬊จ㞟ሢࡢෆ 㒊ࡢ⃰ᗘࡀపࡃ࡞ࡗࡓ⪃࠼ࡽࢀࡿ㸬୍᪉࡛㸪⌫≧ࡔࡗࡓ ⣽⬊จ㞟ሢࡀୖࡽᢲࡉ࠼ࡘࡅࡽࢀࡓࡼ࠺࡞ᙧ≧ኚࡋ ࡚࠸ࡿࡶ⪃࠼ࡽࢀࡿ㸬⣽⬊จ㞟ሢࡣእ࿘ࡢ⣽⬊ࡀ୰ᚰ ᕳࡁ㎸ࡲࢀࡿࡇࡼࡗ࡚จ⦰ࡍࡿࡓࡵ㸪⤒ⓗ⣽⬊ࡀ จ㞟ࡋ࡚࠸ࡃྠ⃰ᗘࡣ㧗ࡃ࡞ࡿ⪃࠼ࡽࢀࡓࡀ㸪⃰ ᗘࡀపࡃ࡞ࡗ࡚࠸ࡿ㒊ศࡶ☜ㄆࡍࡿࡇࡀ࡛ࡁࡓ㸬ࡇࢀࡣ ⣽⬊จ㞟ሢෆ㒊࡛⣽⬊ࡀ⛣ືࡋ࡚࠸ࡿ࡞ࡢኚࡀ⾲ࢀ࡚ ࠸ࡿ⪃࠼ࡽࢀࡿ㸬୍᪉㸪⣽⬊จ㞟ሢࢆస〇ࡍࡿ96 ࢙࢘ࣝ ࣉ࣮ࣞࢺࡣCO2࣮ࣥ࢟ࣗ࣋ࢱ࡛ᇵ㣴ࡉࢀࡿࡓࡵ㸪ྲྀࡾฟ ࡋࡓࡾ⛣ືࡉࡏࡓࡾࡍࡿࡇ࡛㸪⣽⬊จ㞟ሢࡀ࢙࢘ࣝෆ࡛ ື࠸ࡓࡇࡶ⪃࠼ࡽࢀࡿ㸬 ⣽⬊จ㞟ሢ⏬ീࢩ࣮ࣗࢻ࣮࢝ࣛࢆࡍࡇࡼࡗ࡚㸪 ⫗║࡛ࡣุ᩿࡛ࡁ࡞ࡗࡓ㸪⣽⬊จ㞟ሢࡢ⣽⬊ศᕸࢆ☜ㄆ ࡍࡿࡇࡀ࡛ࡁࡓ㸬⥅ⓗ࡞ほᐹࡼࡾ㸪⣽⬊ᩘࡼࡗ࡚ ⣽⬊ࡀจ㞟ࡍࡿ㏿ࡉࡀ␗࡞ࡗࡓࡾ㸪᭱ࡶ⣽⬊ࡀจ㞟ࡋ⌫≧ ࡞ࡿࢱ࣑ࣥࢢࢆ≉ᐃ࡛ࡁࡓࡾࡍࡿࡇࡀ࡛ࡁࡓ㸬ᮏ◊ ✲ࡣ㸪⣽⬊ᵓ㐀యࢆస〇ࡍࡿ㝿ࡸ⣽⬊จ㞟ሢࡢ㠀くホ౯ ࠾࠸࡚㸪᭷⏝࡞ሗࡋ࡚ᐤ࡛ࡁࡿ⪃࠼ࡽࢀࡿ㸬ࡋ ࡋ㸪ࢫࣇ࢙ࣟࢻෆ㒊ࡢ≧ែࡘ࠸࡚ࡣゎᯒ࡛ࡁ࡚࠸࡞ ࠸ࡇࡀᮏㄽᩥࡢ㝈⏺࡛࠶ࡾ㸪ᑗ᮶ⓗࡣ⣽⬊จ㞟ሢࡢᙧ ែࢫࣇ࢙ࣟࢻࡢෆ㒊ࡢ㛵㐃ࡘ࠸࡚ࡶ᫂ࡽࡋ࡚࠸ ࡁࡓ࠸⪃࠼ࡿ㸬
Ώ㒊 ಇᶞ㸪ୗᡞ 4. ⤖ゝ ⣽⬊จ㞟ሢ⏬ീ⃰ᗘศᕸࢆ⾲♧ࡉࡏࡿࢯࣇࢺ࢙࢘ ࡛㸪ࢩ࣮ࣗࢻ࣮࢝ࣛฎ⌮࠾ࡅࡿ⫼ᬒ⃰ᗘࡢᇶ‽್ࢆኚື ࡉࡏࡿࡇ࡛㸪ᚑ᮶ࡢࢯࣇࢺ࢙࡛࢘☜ㄆࡉࢀ࡞ࡗࡓ⣽ ⬊จ㞟ሢෆ㒊ࡢኚࢆ☜ㄆࡍࡿࡇࡀ࡛ࡁࡓ㸬ᚋࡣ㸪 ┦ᕪ㢧ᚤ㙾⤌ࡳྜࢃࡏࡿࡇ࡛㸪⣽⬊จ㞟ሢࡢヲ⣽࡞ศ ᯒࢆ┠ᣦࡋ࡚࠸ࡃ㸬 ㅰ㎡ ᮏ◊✲ࡢ୍㒊ࡣ㸪ᮏᏛሗ⛉Ꮫ◊✲ᡤࡢᖹᡂ29 ᖺᗘⱝᡭ ᩍဨ◊✲㧗ᗘᨭไᗘࡢ◊✲㈝ࡼࡾᐇࡉࢀࡓ㸬ࡇࡇ グࡋㅰពࢆ⾲ࡍࡿ㸬 㸦௧ඖᖺ㸯㸮᭶㸯㸳᪥ཷ㸧 ᩥ ⊩
1㸧 R.Langer, et al.: “Tissue Engineering”, Science, Vol.260-5110, pp.920-92 (1993)
2㸧 M. Harimoto, et al.: J Biomedical Materials Res., 62, 3, 464-470 (2002)
3㸧 M.Nakamura, et al.: Tissue engineering, Vol.11, No.11-12, pp.1658-1666 (2005)
4㸧 V.L.Tsang, A.A.Chen, L.M.Cho, K.D.Jadin, R.L.Sah, S.DeLong, et al.: Fabrication of 3D hepatic tissues by additive photopatterning of cellular hydrogels, The FASEB Journal, Vol.21, No.3, pp.790-801 (2006)
5㸧 A.P.Rago, et al.: Controlling cell position in complex heterotypic 3D microtissues by tissue fusion, Biotechnology and Bioengineering, Vol.102, No.4, pp.1231-1241 (2009) 6㸧 E. D. Miller, et al.: Biomaterials, 27, 10, 2213-2221 (2006) 7㸧 ᑿୖᘯ 㸸⣽⬊ࣇࣂ࣮ࡼࡿ⣽⬊⤌⧊ࡢ㧗ḟᵓ㐀ᵓ ⠏ 㸪 ࣂ ࢜ ࢚ ࣥ ࢪ ࢽ ࣜ ࣥ ࢢ ㅮ ₇ ㅮ ₇ ㄽ ᩥ 㞟 Vol.25㸪 pp.369-370 (2013) 8㸧 ⦾ᐩ㤶⧊㸸⣬ࡢᢡࡾ␚ࡳᢏ⾡ࢆ⏝࠸ࡓ3 ḟඖ❧యᵓ㐀యࡢᵓ ⠏㸪⏕་⒪㸪Vol.10㸪pp.188 (2011) 2㽢104cell/well 3㽢104cell/well
1stday 2ndday 3rdday 4thday 5thday
4㽢104cell/well
Fig.6 Pseudo-color image of spheroids 2㽢104cell/well
3㽢104cell/well
1stday 2ndday 3rdday
4㽢104cell/well
4thday 5thday
