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初代ラット大脳皮質由来神経細胞の凍結保存条件に及ぼす凍結速度
Title Author(s) Citation Issue Date 初代ラット大脳皮質由来神経細胞の凍結保存条件に及ぼ す凍結速度依存性 内田, 努; 本村, 寿太郎; 永山, 昌史; 郷原, 一寿 低温生物工学会誌 = Cryobiology and Cryotechnology, 53(2): 161-166 2007-12-30 DOI Doc URL http://hdl.handle.net/2115/36104 Right Type article (author version) Additional Information File Information uchida-8.pdf Instructions for use Hokkaido University Collection of Scholarly and Academic Papers : HUSCAP Ὼ⏍∸ᕝᏕఌヽࠜCryobiology and Cryotechnologyࠝ㸡 Vol. **, No. *, ***㹳***, 200* ิࣚࢴࢹኬ⬳⓮㈻⏜ᮮ♼⤊ ⣵⬂ࡡ ⤎ಕᏋ᮪௲࡞ཀྵࡌ⤎㏷ ᗐ౪Ꮛ ᛮ ᾇ㐠ኬᏕኬᏕ㝌ᕝᏕ◂✪⛁ හ⏛ ຑ㸡ᮇᮟᑋኯ㑳㸡Ễᒜ᪸ྍ㸡㒋ཋୌᑋ Effects of Cooling Rate on Cryopreservation Conditions of Primary Rat Cortical Cells Tsutomu UCHIDA, Jutaro MOTOMURA, Masafumi NAGAYAMA, and Kazutoshi GOHARA Graduate School of Engineering, Hokkaido University, N13 W8 Kita-ku, Sapporo 060-8628, Japan Developments of cryopreservation techniques on primary rat cortical cells were carried out to observe the effect of cooling rates. Primary rat cortical cells were dispersed in the cryopreserving medium including 5 vol% dimethylsulfoxide, and then they were frozen by various cooling rates ranged from -3 K/min to -150 K/min. The survival ratio was estimated qualitatively by using the phase-contrast microscope. The feasibility of the neural activity was verified by measuring its spontaneous action potential with a multi-electrode array system. Based on these characterizations, the lower cooling rate of the dispersed sample was more feasible for the cryopreservation of the primary rat cortical cells. (Received ***; Accepted ***) ⥬ ゕ ჹ⣌ࡡ୯ᚨ࡛ࡾᚨ➵⣵⬂ኣࡂࡡ⣵⬂⛸࡚㸡⌟ ᅹ࡚ࡵ⤎ಕᏋᢇ⾙ࡡ㛜Ⓠᙽࡒࡿ࡙࠷ࡾ㸣ኣࡂࡡ ⣵⬂ࡷ⤄⧂ࡡ⤎ಕᏋᢇ⾙ࡢ㸡ཿࡂࡢ⏍∸ࡡῺ ⤎ಕᏋྊ⬗⛸㢦ࡡ⣵⬂ࡢቌṢ⬗ࢅ᭯ࡊ࡙࠽ࡽ㸡 㝸ᐐ࡞ᑊࡌࡾ◂✪࡞ጙࡱࡽ㸡⌟ᅹ࡚ࡢ෫㣏ဗ࠾ࡼ ⤎よ࡞࠽࠷࡙΅ᑛࡊࡒ⣵⬂ᩐࡵよᚃࡡᇰ㣬࡞ ්⏍༈⒢࡞㛭ࡌࡾࡵࡡࡱ࡚ᖕᗀࡂ◂✪ࡈࡿ࡙ࡀ࡙࠷ ᑊࡊ࡙ኬࡀᙫ㡢ࢅ࠻ࡾࡆ࡛ࡢ࠷㸣ࡊ࠾ࡊ♼⤊ ࡾࠔཿࡂ࡙᩺ࡊ࠷ၡ㢗ࠕ࡚࠵ࡾ 1) 㸣⏐ᴏ࡞├⤎ࡊࡒ ⣵⬂ࡷᚨ➵⣵⬂ࡢ⣵⬂ฦ࡞ࡻࡾቌṢ⬗࡛ࢆ ၡ㢗࡚࠵ࡾࡒࡴ࡞㸡ᵕࠍࢪࢠ࣭ࣛࢼࣤࢡ࡞ࡻࡖ ↋࠷ࡒࡴ㸡㧏ᗐ⤎ಕᏋᢇ⾙ࡡ㛜Ⓠᚪさ࡛ࡈࡿ ࡙࠵ࡾ⣵⬂⛸࡞ᑊࡌࡾ㐲ว⤎᪁Ἢࢅぜࡗࡄࡾ ࡾࡡ࡚࠵ࡾ㸣 ᢇ⾙㛜Ⓠ࡛ࡊ࡙⾔ࢂࡿ࡙ࡀ࡙࠽ࡽ㸡ࡐࡡ࣒࢜ ິ∸࠾ࡼㄢ⿿ࡈࡿࡒ⣵⬂ࡢ㸡ࡐࡡ⏍∸ᏕⓏ࣒࢜ ࢼࢫ࣑ᇱ♇Ⓩฦ㔕ࡢࡱࡓ༎ฦよ᪺ࡈࡿ࡙࠷ ࢼࢫ࣑ࢅよ᪺ࡌࡾ◂✪࡞࠽࠷࡙ᗀࡂ⏕࠷ࡼࡿ࡙ ࠷ࡡ⌟≟࡚࠵ࡾ㸣ᚉࡖ࡙⤎ಕᏋ࡚ࡀࡾ⣵⬂⛸ࡢ ࠷ࡾ㸣㎾ᖳࡡ⏍⛁Ꮥࡡ◂✪࡚ࡢິ∸モ㥺ࢅᴗງᑛ 㝀ࡼࡿ࡙࠽ࡽ㸡⏍∸ࡡ୯ᯙࢅྒྷࡾ♼⤊⣵⬂ࡷ㸡ᚘ⎌ ࡂࡌࡾ᪁ྡྷ࡚᳠ゞࡈࡿ࡙࠽ࡽ 2) 㸡ⷾࡷ්⏍༈⒢ ฦ㔕࡚ิᇰ㣬⣵⬂ࡡ㞺さᚃቌኬࡌࡾࡆ࡛ ➠ 53 ᅂῺ⏍∸ᕝᏕఌ◂✪ሒ࿈**. ࡈࡿ࡙࠷ࡾ㸣≁࡞♼⤊⣵⬂ࡡ⤎ಕᏋࡢᏕ⾙ [Key words: Neuron, Cryopreservation, Cooling 㟻㸡⏐ᴏ㟻࡞࠽࠷࡙㔔さ࡚࠵ࡾ㸣ౚ࠻ࡣ㸡♼⤊⣵⬂ rate, Action potential; ♼⤊⣵⬂㸡⤎ಕᏋ㸡⤎ ࡢᑛ㔖ࡡẐᛮ∸㈻࡞ᑊࡊ࡙ࡵཬᚺࢅ♟ࡌࡆ࡛࠾ࡼ㸡 ㏷ᗐ㸡Ὡິ㞹న] ⷾࡡࢪࢠ࣭ࣛࢼࣤࢡ࡞⏕࠷ࡾࡆ࡛ࡢ㟸ᖏ࡞᭯ຝ ࡚࠵ࡾ࡛⩻࠻ࡼࡿ࡙࠷ࡾ㸣ᚉࡖ࡙ࡆ࠹ࡊࡒ㞺さࡡ㧏 ࠷࠹ᶭ⬗ಕᏋࡈࡿࡄࡿࡣࡼ࠷㸣ࡊ࠾ࡊ㸡⌟ ࠷⣵⬂ࢅ⤎ಕᏋࡌࡾᢇ⾙㛜Ⓠࡢ㸡ୠ⏲୯ࡡ㞺さ ᅹࡱ࡚ࡐ࠹ࡊࡒ⤎ಕᏋ࣬ဗ㈻フ౮ᢇ⾙☔❟ࡊ࡙ ࡞ᑊࡌࡾᆍ㈻⣵⬂ࡡᏭᏽ౩⤝࡞࡛ࡖ࡙㟸ᖏ࡞㔔さ ࠷ࡾ࡛ࡢゕ࠷㞬࠷㸣 ࡚࠵ࡾ㸣⤎ࡌࡾฺⅤ࡛ࡊ࡙㸡ㄢ⿿ࡊࡒิ⣵⬂ࢅ ᮇ◂✪ࡢ㸡ฦᩋࡈࡿࡒิ⣵⬂ࢅࡐࡡᶭ⬗ࢅ ࢤࣤࢰ࣐ࢾ࣭ࢨࣘࣤ(⣵⬂ࡡỗ)ࡈࡎࡍ࡞㛏㛣ಕ ࢂࡍ࡞⤎ಕᏋࡌࡾᢇ⾙ࡡ㛜Ⓠ࡛࣒࢜ࢼࢫ࣑ࡡよ᪺ Ꮛ࡚ࡀࡾࡆ࡛ࡷ㸡ᚪさ࡞ᚪさ㔖ࡓࡄཱིࡽฝࡊ࡙ ࢅ┘Ⓩ࡛ࡊ࡙࠷ࡾ㸣≁࡞㞺さࡡ㧏࠷♼⤊⣵⬂ࡷᚨ➵ ⏕࡚ࡀࡾࡆ࡛ᣪࡅࡼࡿࡾ㸣ࡱࡒ㸡⤎࡞ ⣵⬂ࢅᑊ㇗࡞㸡⤎㏷ᗐ࡛⤎ಕ㆜࡞ࡗ࠷࡙Ἰ┘ ࡢ⣵⬂ࡡㅨᴗ❻࡞ᢒ࠻ࡼࡿࡾࡡ࡚㸡⏍∸࡞࡛ࡖ ࡊ࡙◂✪ࢅ㐅ࡴ࡙࠷ࡾ ࡙ࡡ㛣ࢅḾࡴࡼࡿࡾ࠾ࡵࡊࡿ࠷࡛࠷࠹⮾ࢅᢢ ᑊ㇗࡞㸡⤎㏷ᗐࡡ㐢࠷⣵⬂ࡡ⸵⏍⋙࡞ཀྵࡌᙫ 3) ࡂࡆ࡛ࡵ࡚ࡀࡾ 㸣 9) 㸣ࡆࡆ࡚ࡢิ♼⤊⣵⬂ࢅ 㡢ࢅ᳠ゞࡊࡒ㸣ࡈࡼ࡞よࡊࡒ⣵⬂ࢅኣ㞹ᴗᇱᯀ୕ ⣵⬂ࢅ⤎ࡌࡾ㝷࡞ࡢ㸡⣵⬂හࡡểᬏ⏍ᠺࢅᢒโ ࡌࡾࡆ࡛㔔さ࡚࠵ࡾ㸣⤎よ࡞ࡻࡖ࡙⣵⬂ ഭࢅུࡄࡾさᅄࡢ࠷ࡂࡗ࠾⩻࠻ࡼࡿ࡙࠷ࡾ 1, 4) ࡚ᇰ㣬ࡌࡾࡆ࡛࡚㸡ྜྷ࡞ኣᩐࡡ⣵⬂ࡡᶭ⬗ࢅ⠾౼ ࡞ㄢࡾフ౮Ἢࢅ㛜Ⓠࡊࡒ㸣 㸣ౚ࠻ ᮞᩩ࠽ࡻࡦ᪁Ἢ ࡣ㸡⣵⬂හ࡞⏍ᠺࡊࡒể⤎ᬏ⣵⬂හ㒂ࡡ々㞟ᵋ ㏸మ࡞∸⌦Ⓩᙫ㡢ࢅཀྵࡌࡆ࡛ᣪࡅࡼࡿࡾ㸣ࡱ ࡒ㸡⣵⬂አ࡞⏍ᠺࡊࡒể⤎ᬏ⣵⬂⭯࡞∸⌦Ⓩᙫ ࡱࡍ⤎᪁Ἢࡡ㐢࠷࡞ࡻࡾ⤎㏷ᗐࢅぜ✒ࡵࡾࡒ 㡢ࢅ࠻ࡒࡽ㸡⣵⬂አࡡể⤎ᬏᠺ㛏࡞ఔࡖ࡙⣵⬂හ ࡴ㸡⣵⬂⤎࡞⏕࠷ࡾᇰ㣬ᾦࢅࢠ࢛ࣚࢲ࣭ࣖࣇ୯ 㒂ࡡ⁈ᾦ⃨⦨ࡈࡿࡾᾈ㏩ᅸࢨࣘࢴࢠࡡࡻ࠹Ꮥ ࡞ 1ml ථࡿࡒࡵࡡࢅモᩩ࡛ࡊ࡙㸡⇍㞹ᑊ࡞ࡻࡾῺᗐ Ⓩᙫ㡢ࡵ࠵ࡾ 4, 5) 㸣ể⤎ᬏ⏍ᠺࡈࡿࡾሔᡜ࡞ࡗ࠷ ゝῼࢅ⾔ࡖࡒ㸣⠾༟෫༴᪁Ἢ࡚෫༴㏷ᗐࢅᗀ⠂ᅑ ࡙ࡢ㸡⤎ࡌࡾ㝷ࡡ෫༴㏷ᗐࡡ㐢࠷࡞ࡻࡖ࡙࠵ࡾ⛤ ࡞ንࡈࡎࡾࡒࡴ㸡ᾦమ✽⣪ཧࡢ㸢80ΥࡡῺᗐࢅಕ 4, 6) 㸣᛬㏷⤎ 㸝⤎㏷ᗐ㸯ᩐⓊΥ/min ⛤ ࡙ࡾ Deep freezer ࢅ⏕࠷࡙⤎ࡈࡎࡒ㸝ձDeep freezer ᗐ㸞࡛⥾ះ⤎㸝⤎㏷ᗐ㸯1Υ/min ⛤ᗐ㸞ࡡ୦⩽ ୯᩷⇍ᮞහᨲ⨠㸡ղDeep freezer ୯ᨲ⨠㸡ճᾦమ✽⣪ ࡞࠽࠷࡙㸡⤎㐛⛤࡚⏍ᠺࡌࡾể⤎ᬏ࡞ࡻࡽ⣵⬂ࡢ ᾦ㟻├୕ಕᣚ㸡մᾦమ✽⣪୯ᾈ₍㸞㸣ࡐࡡ⤎ᯕ㸡⤎ ୕オࡡࡻ࠹ᙫ㡢ࢅུࡄࡾ㸣ࡊ࠾ࡊ㸡᛬㏷⤎࡚ࡢ ㏷ᗐࡢࡐࡿࡑࡿ Table 1 ࡡࡻ࠹࡞ࡽ㸡෫༴㏷ᗐ 2 ⣵⬂හࡱ࡚⤎ࡊ࡙࠷ࡾࡡ࡞ᑊࡊ࡙⥾ះ⤎࡚ࡢ⣵ ᰾␏ࡾᡥἪࢅᚋࡾࡆ࡛࡚ࡀࡾࡆ࡛☔ヾࡈࡿࡒ㸣 ⬂አࡡࡲࡖ࡙࠷ࡾࡻ࠹࡞㸡⤎㏷ᗐ␏ࡾ࡛ ࠽ಕᏋ㛣ࡢ㸡᪁Ἢ࡚⏕࠷ࡒ෫፳ࡡῺᗐ࡚ಕᣚ ⣵⬂ུࡄࡾể⤎ᬏࡡᙫ㡢ࡵንࡌࡾ㸣ࡱࡒ⣵⬂ ࡊࡒ㸣 ᗐโᚒ࡚ࡀࡾ ⤎ࡡ㝷࡞ࡢ㏳ᖏ㸡⤎よ࡞ࡻࡾࢪࢹࣝࢪࢅ㍇΅ࡊ ࡙⣵⬂⸵⏍⋙ࢅྡྷ୕ࡈࡎࡾࡒࡴ㸡⤎ಕ㆜࡛ࡣ ࡿࡾ∸㈻῟ຊࡈࡿࡾ㸣ࡆࡿࡱ࡚ࢡࣛࢬࣛࣤ 㸝 glycerol 㸞 ࡷ ࢩ ࣒ ࢲ ࣜ ࢪ ࣜ ࣆ ࢚ ࢞ ࢨ ࢺ Table 1. Cooling rate and cryopreservation temperature for each freezing process Freezing procedures Cooling rates ձ stored in the insulation box in the deep freezer ղ stored in the deep freezer ճ kept at just above liquid N2 pool մ kept in liquid N2 pool 㸢3Υ/min Storage temperatures 㸢80Υ 㸢10Υ/min 㸢80Υ 㸢30Υ/min 㸢197Υ 㸢150Υ/min 㸢197Υ 㸝dimethylsulfoxide, DMSO㸞ኣࡂࡡ∸㈻⤎ಕ ㆜࡛ࡊ࡙⏕࠷ࡼࡿ࡙ࡀࡒ 7) 㸣ࡐࡊ࡙㸡᭯ຝಕ㆜ ࡡ⛸㢦ࡢ⣵⬂⛸࡞ࡻࡖ࡙ࡵ␏ࡾࡆ࡛ฦ࠾ࡖ࡙ ࠷ࡾ 8)㸣 ㏳ᖏ⣵⬂ࡡ⤎ಕᏋᢇ⾙ࡡྊྫྷࡢ㸡࡛ࡊ࡙よ ᚃࡡ⣵⬂ࡡ⸵⏍⋙࡚フ౮ࡈࡿ࡙࠷ࡾ㸣ࡱࡒ⣵⬂ᶭ⬗ ࡡフ౮Ἢ࡛ࡊ࡙㸡⣵⬂ࢅᇰ㣬࣬ฦࡈࡎ≁␏ࢰࣤࣂ ࢠ㈻ࡡⓆ⌟➴ࢅㄢࡾ᪁Ἢ࠵ࡾ㸡フ౮ࡱ࡚㛏 㛣࠾࠾ࡽ㸡ࡐࡡ୕⣵⬂⮤㌗ࡵഭࡗࡄ࡙ࡊࡱ࠹ḖⅤ ࠵ࡖࡒ㸣ࡈࡼ࡞♼⤊⣵⬂ࡷᚨ➵⣵⬂➴ࡡ㞹ẴⓏὩິ ⤎ᐁ㥺࡞⏕࠷ࡒモᩩࡢ㸡SD ࣚࢴࢹ᩺⏍ඡ㸝1㹳3 ࢅ⾔࠹ิᇰ㣬⣵⬂ࡢ㸡よᚃ࡞࣭ࣗࢧ࣭ࡡ⏕ࡌ ௦㸞ࡡኬ⬳⓮㈻⏜ᮮࡡิ♼⤊⣵⬂࡚࠵ࡾ㸣ᇰ㣬 ࡾモ㥺ᶭ࡚ᇰ㣬࡚ࡀ㸡⮤ⓆⓏ࡞㞹ẴⓏὩິࢅ⾔࠹࡛ ᾦࡢ DMEM (Dulbecco’s modified Eagle’s medium) +10vol%ࢨ⫶ඡ⾉ΰ㸝fetal bovine serum㸯FBS㸞ࢅ ⏕࠷ࡒ㸣ᮇ◂✪࡚⏕࠷ࡒิ♼⤊⣵⬂࠽ࡻࡦᇰ㣬ᾦ ᶭ⬗ࢅⓆᥱࡌࡾࡆ࡛ࢅ☔࠾ࡴࡾࡒࡴ㸡ᠩ⃦ᾦࡡୌ㒂 ࡢ㸡 㸝ᰬ㸞ࣈ࣏࣭ࣚࣛࢬࣜࡻࡽᥞ౩ࡈࡿࡒ㸣ࡆࡡᇰ ࢅ⤎ࡈࡎࡍ࡞ᇰ㣬ࡊࡒࡵࡡ࡞ࡗ࠷࡙㸡⣵⬂╌ࡡ 㣬ᾦ࡞⤎ಕ㆜㸝5vol㸚DMSO㸞ࢅຊ࠻ࡒ⤎⏕ ᵕᏄࡷ⮤ⓆⓆℾࡡ☔ヾࢅᑊ↯ᐁ㥺࡛ࡊ࡙⾔ࡖࡒ㸣 ᇰ㣬ᾦ࡞㸡⣵⬂ᐠᗐ 1×106 cells/ml ࡛ࡾࡻ࠹࡞ฦ ᩋㄢᩒࡊᠩ⃦ࡈࡎࡒ㸣ࡆࡡᠩ⃦ᾦ 1 ml ࢅࢠ࢛ࣚࢲ ⤎ᯕ࠽ࡻࡦ⩻ᐳ ࣭ࣖࣇ࡞ථࡿ㸡୕オࡡࡻ࠹ᵕࠍ⤎᪁Ἢ࡚⤎ ࡊࡒ㸣ࡐࡊ࡙㸢80Υ㸡ཧࡢ㸢197Υ࡚ 1 㐄㛣ಕᏋࡊࡒ ᚃ㸡37Υ࡚ ᮅ⤎ࡡิ♼⤊⣵⬂ࡡᇰ㣬ᐁ㥺࡚ふᐳࡌࡾ࡛㸡 ↞ࡊ࡙᛬㏷よࡊࡒ㸣よᚃ⤎ಕ㆜ ᇰ㣬㛜ጙ࡞ࡢධ࡙⌣≟࡚ᾃ㐗ࡊ࡙࠷ࡒ⣵⬂㸡ᩐ ⣵⬂࡞ᙫ㡢ࢅཀྵࡈࡻ࠹㸡㏷ࡷ࠾࡞㏳ᖏࡡᇰ 㛣㹳ᩐ࡚ᇱᯀ୕࡞╌ࡊ㸡✲㉫ࢅఘࡣࡊ࡙ᠺ㛏 㣬ᾦ࡞⨠ᥦࡊࡒ㸣࠽㸡DMSO ࢅຊ࠻ࡍ࡞⤎ࡈࡎ ࡊ࡙࠷ࡾᵕᏄふᐳࡈࡿࡒ㸣ࡱࡒ⣵⬂አ㞹నῼᏽࢅ ࡒᑊ↯ᐁ㥺ࡵ⾔ࡖࡒ㸝෫༴᪁Ἢࡢղ㸞㸣 ⾔ࡖࡒ⤎ᯕ㸡ᇰ㣬㛜ጙᚃ 2 㐄㛣⛤ᗐ࡚ⓆℾὩິࢅ㛜 よᚃࡡ♼⤊⣵⬂ࡢ㸡⣵⬂ᇰ㣬⏕࢘ࣜ୕࡚Ὼᗐ ጙࡊࡒ㸣ᮅ⤎⣵⬂ࡡὩິ㞹నࡢ㸡ᣲᖕ 34.2s3.7 µV㸡 37Υ㸡ᗐ100㸚㸡CO2ࢅ5㸚ྱ᭯ࡊࡒ1Ẵᅸࡡ✭Ẵࡡ ᖕ 2.3s0.5 ms ⛤ᗐ࡛ῼᏽࡈࡿࡒ㸣ㄏᕣࡢὩິ㞹న ⎌ሾୖ࡚ῺᗐࡷS-ࢅୌᏽ࡞ಕࡖ࡙ᇰ㣬ࡊࡒ㸣ࡐࡊ࡙ ῼᏽࡈࡿࡒ⣑ 20 ࡡ㞹ᴗ࡚ࡡᖲᆍೋ࠾ࡼࡡ᭩ኬ೩ ࡐࡡ⸵⏍≟Ἓࡢ㸡ᇰ㣬㛜ጙᚃࡡᙟឺንࢅన┞ᕣ㢟 ᕣࢅ⾪ࡌ㸣ࡆࡡ㞹నࡢ⣵⬂አオ㘋Ἢࢅ⏕࠷࡙ῼᏽࡈ ᚜㙶࡚ふᐳࡊ࡙⾔ࡖࡒ㸣╌࣬ఘᘿࡊࡒ⣵⬂ࡢよ ࡿࡾᣲᖕ ᚃ⸵⏍ࡊࡒ⣵⬂࡚࠵ࡾ࡛⩻࠻㸡ᮅ⤎࡚╌ࡊࡒ ms ࡡ࢛࣭ࢱ࣭࡚࠵ࡾࡆ࡛࠾ࡼ♼⤊⣵⬂ࡡὩິ㞹న ⣵⬂㟻✒࡞ᑊࡌࡾ⤎ᚃ⸵⏍࣬╌ࡊࡒ⣵⬂㟻✒࡞ ࡚࠵ࡾ࡛ึ᩷ࡊࡒ㸣ࡐࡆ࡚㸡ࡆࡡೋࢅᮇ◂✪࡚⏕࠷ ࡻࡖ࡙㸡⸵⏍ࡡ⛤ᗐࢅᏽᛮⓏ࡞フ౮ࡊࡒ㸣 ࡒ♼⤊⣵⬂ࡡὩິ㞹నࡡᇱ‵ೋ࡛ࡊࡒ㸣 ࡱࡒ⸵⏍ࡊࡒ♼⤊⣵⬂ࡡᶭ⬗ࡡḿᖏᛮࢅフ౮ࡌࡾ 12, 13) ࡛ྜྷ⛤ᗐ࡚㸡Ὡິ㞹నࡡಘྒᖕࡵᩐ 䐖 䐗 ࡒࡴ㸡ᇰ㣬ᇱᯀ୕࡞Ⅴࡡ᚜ᑚ㞹ᴗࢅ᭯ࡌࡾࢨ࣭ࣔ ࣝ㸝ኣ㞹ᴗᇰ㣬ᇱᯀ㸞୕࡞⣵⬂ࢅᇰ㣬ࡊ㸡ࡐࡡ⮤Ⓠ Ⓠℾࢅゝῼࡊࡒ㸣ࡆࡡ᪁Ἢࡢ㸡♼⤊⣵⬂ࡡ⣵⬂አὩ ິ㞹నࢅゝῼࡌࡾᡥἪࡡࡥ࡛ࡗ࡚࠵ࡾ㸣⣵⬂አオ 㘋Ἢ࡚ࡢ㸡⣵⬂አ࡚ࡡ࢛ࣤࡡ㔖Ⓩንᘤࡀ㉫ࡆ 100µm 䐘 䐙 ࡌ㞹నᕣࢅオ㘋ࡌࡾࡡ࡚㸡ᚋࡼࡿࡾ㞹నᣲᖕࡢ⣵⬂ හオ㘋Ἢࡡᩐ༎P;ࡻࡽᑚࡈೋ㸡㏳ᖏᩐ༎µ;ࡡ࢛ ࣭ࢱ࣭࡛ࡾ㸡㸣ࡐࡡࡒࡴ㸡ゝῼ⣌ࡡࢿࢫࣝ࣊ ࣜࡵ㹳µ;⛤ᗐ࡞ᢒ࠻ࡒ㸣ᮇ◂✪࡚⏕࠷ࡒ⣵⬂ ࡡὩິ㞹నࢅῼᏽࡌࡾࢨ࣭ࣔࣝ࡞ࡢ㸡࢝ࣚࢪᇱᯀ୕ ࡡ୯ኳ㒂࡞ୌ㎰µPࡡᄿみ࠷Ⓣ㔘㯦㞹ᴗᇔ ࡴ㎲ࡱࡿ㸡ࡱࡒࢨ࣭ࣔࣝࡡ୯ᚨ࠾ࡼ༎ฦ㞫ࡿࡒ࿔㎰ 㒂࡞ࡢࡡᇱ‵㞹ᴗス⨠ࡈࡿ࡙࠷ࡾ㸣ࡆࡡࢨࣔ ࣭ࣝ୯ኳ㒂࡞⣵⬂ࢅᇰ㣬ࡌࡾࡆ࡛࡚㸡ᶭⓏഭ ࢅ࠻ࡾࡆ࡛ࡂὩິ㞹నࡡῼᏽ࡚ࡀࡾ㸣⣵⬂ࡡ ╌ࢅಀ㐅ࡌࡾࡒࡴ㸡ࢨ࣭ࣔࣝ୕࡞ࢤ࣭ࣚࢣࣤࡱࡒ Fig. 1. phase-contrast images of neurons at 9 days after culture. These images represent typical morphology of cells cryopreserved in the following 4 cooling rates; ձ㸢3Υ/min, ղ㸢 10 Υ /min, ճ 㸢 30 Υ /min, մ 㸢 150 Υ /min. Cells unable to adhere to substrate form cell aggregations (arrows in ճ and մ). ࡢSRO^1O^VLQHࢅࡂࢤ࣭ࢷࣤࢡࡊࡒ㸣♼ ձ㹳մࡡ᪁Ἢ࡚⤎ಕᏋࡊࡒモᩩࡡ㸡よᚃ 9 ⤊⣵⬂ࡡὩິ㞹నࡡῼᏽ࡞ࡢ㸡2*)8^VWHP㸝ࣜ ┘ࡡᇰ㣬≟Ἓࢅ Fig. 1 ࡞♟ࡌ㸣⤎よᚃࡡ⣵⬂ ࣆ࣒ࢴࢺࢦ࢙ࣤࢪ♣⿿㸞ࢅ⏕࠷ࡒ㸣ࡱࡒ♼⤊⣵ ࡚ࡢ㸡ᮅ⤎ࡡࡵࡡ࡛Ẓ࡙ᇱᯀ࡞㈖ࡽࡄ࠷⣵ ⬂ࡡὩິ㞹నࡡಘྒᖕࡢᩐPVࡡ࢛࣭ࢱ࣭࡚࠵ࡾࡡ࡚㸡 ⬂㸝Fig.㸦࡞࠽࠷࡙࿔㎰㒂Ⓣࡂකࡖ࡙࠷ࡾ୷࠷⣵ ࡆࡡ⨠࡚ࡢN-_࡚ῼᏽࢅ⾔ࡖࡒ㸣 ⬂㸞ࡡྙኣ࠷ࡆ࡛ふῼࡈࡿࡒ㸣⤎ಕᏋྊ ⏕࠷ࡒิ♼⤊⣵⬂ᮇ◂✪ࡡᇰ㣬᮪௲࡚ḿᖏ࡞ ⬗࡚࠵ࡾ⣵⬂࡚ࡵ㸡⤎࣬よࣈࣞࢬࢪࡡ୯࡚ᩐ 10㸚⛤ᗐ⸵⏍࡚ࡀ࠷ࡆ࡛ሒ࿈ࡈࡿ࡙࠷ࡾ 14)ࡒࡴ㸡 ḗ࡞㸡⸵⏍ࡊࡒ♼⤊⣵⬂ࡡᶭ⬗ࡡḿᖏᛮࢅ☔ヾࡌ ᮇ◂✪࡚⏕࠷ࡒࣈࣞࢬࢪ࡞࠽࠷࡙ࡵ㸡♼⤊⣵⬂࡞ర ࡾࡒࡴ㸡Ὡິ㞹నࡡῼᏽࢅ⾔ࡖࡒ㸣ୌౚ࡛ࡊ࡙ Fig. 2 ࡼ࠾ࡡഭࢅཀྵࡊࡒࡵࡡ࡛⩻࠻ࡼࡿࡾ㸣ࡌࢂࡔ㸡 ࡞ࡢኣ㞹ᴗᇰ㣬ᇱᯀ୕࡞ᇰ㣬ࡊࡒ㸡⤎よࡈࡿࡒ ࡆࡡ⸵⏍⋙ࡡୖࢅᑛࡊ࡚ࡵᑚࡈࡂࡌࡾࡆ࡛㸡ᮇ ♼⤊⣵⬂ࡡన┞ᕣ㢟᚜㙶ാࢅ♟ࡌ㸝⤎᪁Ἢձ࡞ࡻ ◂✪ࡡ┘Ⓩ࡚࠵ࡾ㸣ࡆࡿࡼ╌࡚ࡀ࠾ࡖࡒ⣵⬂ࡢ㸡 ࡾ⤎ಕᏋ⣵⬂ࡡ㸡よᚃᇰ㣬 16 ┘ࡡᵕᏄ㸞㸣ࡆ ᇰ㣬 1 㐄㛣⛤ᗐ࡚ም≟࡞ࡽ㏩᪺࡞ࡾ㸝Fig. 1 ࡡᅒ୯㸡ළ࡚♟ࡊࡒ㞹ᴗ࡚ῼᏽࡈࡿࡒὩິ㞹నࢅ㸡 ճ㸡մ୯▦༰࡚♟ࡈࡿࡒ≟ឺ㸞 㸣ᇱᯀ࡞╌ࡊ࡙࠷ࡾ Fig. 3 ࡞♟ࡌ㸣ࡆࡡ࡛ࡀῼᏽࡈࡿࡒὩິ㞹నࡢ㸡ᖲ ⣵⬂ࡵ㸡ḿᖏ࡞⸵⏍ࡊ࡙࠷ࡿࡣఘ㛏ࡌࡾ㸡ࡐ࠹࡚ ᆍࡌࡾ࡛ᣲᖕ 23.5s2.1 µV㸡ᖕ 2.3s0.5 ms ࡚࠵ࡽ㸡 ࠷∸ࡢ⌣≟ࡡࡱࡱ࡚࠵ࡾ㸣௧୕ࡡࡻ࠹㸡⤎よ ᮅ⤎⣵⬂ࡡὩິ㞹న࡛Ẓ㍉ࡊ࡙ᣲᖕⱕᖱᑚࡈࡂ ᚃᇰ㣬ࡊࡒ♼⤊⣵⬂ࡡᙟឺふῼ࠾ࡼ㸡⤎ಕᏋ ࡖ࡙࠷ࡒ㸡ୌฦ㛣ࡡᖲᆍⓆℾᅂᩐ⣑ 600 ᅂ ᪁Ἢ࡞ࡻࡾ⣵⬂ࡡ⸵⏍ࡡ⛤ᗐࢅᏽᛮⓏ࡞ぜ✒ࡵࡖࡒ㸣 /min ࡛༎ฦὩⓆ࡞Ὡິࡊ࡙࠷ࡾࡆ࡛♟ࡈࡿࡒ㸣 ࡐࡡ⤎ᯕ㸡᭩ࡵ⸵⏍ࡡ⛤ᗐ㧏࠾ࡖࡒ⤎᪁Ἢࡢձ ࠽ࡆࡿࡼࡡೋࡡ㐢࠷⤎ಕᏋ࡞ࡻࡾᙫ㡢࠾࠹࠾ ࡚㸡࠾ࡖࡒࡡࡢմ࡚࠵ࡖࡒ㸣ࡌࢂࡔ♼⤊⣵⬂ࡢ㸡 ࡞ࡗ࠷࡙ࡢ㸡ᇰ㣬ᩐࡷ⣵⬂ᐠᗐ༎ฦโᚒ࡚ ⤎㏷ᗐ㐔࠷㧏࠷ྙ࡚⸵⏍ࡊࡒࡆ࡛ࢂ࠾ ࡀ࡙࠽ࡼࡍ㸡࠽࠾ࡗ⣵⬂አ㞹న࡞ࡻࡾῼᏽࡡࡒࡴ ࡖࡒ㸣ࡱࡒᇰ㣬ࡡ♼⤊⣵⬂ࡡᙟឺንࡢ㸡⸵⏍⋙ ᏽ㔖ⓏẒ㍉ࡢ࡚ࡀ࡙࠷࠷㸣 ࡡ㧏࠷᮪௲࡚⤎ࡊࡒሔྙࡢᑊ↯ᐁ㥺࡚࠵ࡾᮅ⤎ ⣵⬂ࡡᙟឺን࡞㟸ᖏ࡞ࡻࡂజ࡙࠷ࡒ㸣ࡆࡡࡆ࡛࠾ 50µm ࡼࡵ㸡⸵⏍⋙ࡡ㧏࠷᮪௲࡞ࡻࡾ⤎ಕᏋ⣵⬂ࡡḿᖏ ᛮࡡ㧏ࡈᨥᣚࡈࡿࡾ㸣 ࠽ղࡡ᪁Ἢ࡚⤎ಕᏋࡊࡒ⣵⬂࡞ࡗ࠷࡙㸡ᇰ㣬 㸫 ┘ ࡞ ♼ ⤊ ⣵ ⬂ ≁ ␏ ࢰ ࣤ ࣂ ࢠ ㈻ 㸝 microtuble associated protein 2: MAP2㸞ࡡ⺧කⰅࢅ⾔ࡖࡒ 9)㸣 ࡐࡡ⤎ᯕ㸡⣵㛏࠷✲㉫ࢅఘࡣࡊࡒ⣵⬂ࡡኣࡂ♼⤊ ⣵⬂࡚࠵ࡾࡆ࡛☔ヾࡈࡿࡒ㸣ࡐࡡࡡ⣵⬂ࡢ㸡ࢡ ࣛ⣵⬂ቌṢ⬗ࡡ㧏࠷⣵⬂࡚࠵ࡾ࡛⩻࠻ࡼࡿࡾ㸣 ࡱࡒಕᏋῺᗐ࡞ࡗ࠷࡙ࡢ㸡㸢80Υ࡞࠽࠷࡙ࡵ࠵ࡾ ⛤ᗐࡡᾦమ≟ឺࡡỀᏋᅹࡊ㸡㛏ಕᏋࡌࡾ㛣࡞⏍ Ꮥཬᚺࡹࡖࡂࡽ㐅⾔ࡊࡒࡽ㸡㐛࡞⬲Ềࡈࡿࡒ ࡽࡊ࡙㸡⸵⏍⋙ୖࡌࡾ࡛࠷ࢂࡿ࡙࠷ࡾ 8, 15) 㸣ᮇ ◂✪࡚ࡢ౼୕෫༴᪁Ἢ࡞ࡻࡽಕᏋῺᗐ␏ࡖ࡙ ࠷ࡾ㸡ಕᏋ㛣 1 㐄㛣⛤ᗐ࡛Ẓ㍉Ⓩ▯࠷ࡒࡴ㸡 ࡆࡡࡻ࠹ಕᏋ㛣୯࡞㉫ࡆࡾཬᚺࡡᙫ㡢ヾࡴࡼ ࡿ࠷⛤ᗐᑚࡈ࠷࡛⩻࠻ࡼࡿࡾ㸣ࡊ࠾ࡊ㸡ࡐࡡ㐢࠷ ࡞ࡻࡾ⸵⏍⋙ࡡᙫ㡢࡞ࡗ࠷࡙ࡢᚃࡡㄚ㢗࡚࠵ࡾ㸣 ࡔࡲ࡞⤎ಕ㆜ࢅຊ࠻ࡍ࡞⤎ࡊࡒモᩩ࡞ࡗ ࠷࡙ࡢ㸡࡛ࢆ╌ࡌࡾ⣵⬂ࡢࡲࡼࡿ࠾ࡖࡒ㸣 ࡆࡿࡢ㸡⤎よࡡ㐛⛤࡚ഭࡊࡒ࠾㸡ᡀ࠷ࡢ⸵⏍ Fig. 2. phase-contrast images of neuronal cells cultured on the multi-array electrode. The cells were cryopreserved in the condition of ձ , and then were thawed. Dark 50 µm squares correspond to electrodes. Neurons show spherical cell shape with diameter of approximately 10 µm. Action potential measured by the electrode (marked by circle) is shown in Fig. 3. ࡊࡒᇱᯀ࡞ᑊࡌࡾ╌⬗ງᘽࡂࡖࡒࡵࡡࡓ࡛ ⤎よࡈࡿࡒ♼⤊⣵⬂ࡡ⤎᪁Ἢࡡ㐢࠷࡞ࡻࡾ ᛦࢂࡿࡾ㸣ᇰ㣬ᾦ୯࡞ࡢ 10vol㸚FBS ྱࡱࡿ࡙࠷ ⮤ⓆⓆℾࡡᵕᏄࡵふᐳᐁ㥺ࡡ⤎ᯕ࡛ᩒྙࡊ࡙࠽ࡽ㸡 ࡾ㸡ᮇ◂✪ࡡᐁ㥺᮪௲ࡡ⠂ᅑහ࡚ࡢ 10vol㸚⃨ᗐࡡ ձ㸡ղ࡚ࡢᇰ㣬 2 㐄㛣┘௧㜾࡞⮤ⓆⓆℾ☔ヾࡈࡿ FBS ࡢ⤎ಕ㆜ష⏕ࢅⓆᥱࡊ࠾ࡖࡒ㸡࡛⤎ㄵ࡚ࡀ ࡒ㸣ࡆࡿࡼࡡ♼⤊⣵⬂࡚ᚋࡼࡿࡒὩິ㞹నࡡᙁᗐࡵ ࡾ㸣⾉ΰࡡ⤎ಕ㆜ష⏕ࡢ㸡ࡻࡽ㧏⃨ᗐࡡ῟ຊᚪ 㢎ᗐࡵ㸡ᑊ↯ᐁ㥺࡚࠵ࡾᮅ⤎⣵⬂ࡡࡵࡡ࡞༆ᩓࡊ さࡡ࠾ࡵࡊࡿ࠷ 15) 㸣 ࡙࠽ࡽ㸡⣵⬂ࡡḿᖏᛮ♟ြࡈࡿࡾ㸣ճ࡞ࡗ࠷࡙ࡢ㸡 ⮤ⓆⓆℾ࡛ᛦࢂࡿࡾಘྒྜྷ࡞᳠ฝ࡚ࡀࡒ ࡌຝᯕ࡛ࡊ࡙ࡢ㸡ฆᅖⅤ㜾ୖ࡞ࡻࡽể⤎ᬏ⏍ᠺࡡ 㸡ᙁᗐࡵ㢎ᗐࡵ༎ฦ࡚ࡢ࠾ࡖࡒ㸣ࡆࡿࡢࠉḿᖏ 㔖ࡷ㏷ᗐࢅᢒโࡊ㸡⣵⬂හ࠾ࡼࡡ㐛⬲Ềࡷ⣵⬂ ࡞ᶭ⬗ࢅⓆᥱ࡚ࡀࡾࡻ࠹♼⤊⣵⬂ࡡ⸵⏍⋙࠾ አể⤎ᬏ࡞ࡻࡾᶭⓏഭࢅ㍇΅ࡌࡾ࡛ࡈࡿ࡙ ࡖࡒࡆ࡛ࢅ♟ြࡌࡾࠊմ࡞ࡗ࠷࡙ࡢ㸡㞹ᴗᇱᯀ࡞ᘿ ࠷ࡾ 4)㸣ࡆࡿ࡞ຊ࠻࡙ DMSO ࡞ࡻࡾ⣵⬂⤎ࡡಕ ࡽࡂࡆ࡛ࡡ࡚ࡀࡒ⣵⬂⮤మᑛࡂ㸡⮤ⓆⓆℾࡢ ㆜࣒࢜ࢼࢫ࣑ࡢ㸡⣵⬂හࡡỀ࡛ DMSO ⨠ࡀᥦࢂ ᳠ฝࡈࡿ࠾ࡖࡒ㸣 ࡾࡆ࡛࡞ࡻࡖ࡙⤎ࡡ㝷࡞࠽ࡆࡾ⣵⬂හࡡ⁈ᾦ⃨⦨ ࢅ㍇΅ࡌࡾ㸡࠵ࡾ࠷ࡢ DMSO ⣵⬂⭯ࡡỀᛮࡡ 10µV 5 sec 㒂ฦ࡞⏻ࡱࡖ࡙⭯ࢅᏭᏽࡈࡎࡾࡒࡴ࡛⩻࠻ࡼࡿ࡙ ࠷ࡾ 4, 16)㸣ᮇ◂✪࡚⤎㏷ᗐ౪Ꮛᛮࢅ᳠ゞࡊࡒ⤎ᯕ㸡 A ⤎㏷ᗐ㐔࠷⸵⏍⋙㧏࠾ࡖࡒ㸣ᚉࡖ࡙㸡᛬ ㏷⤎ࡊࡒモᩩ࡞࠽࠷࡙ࡢ㐛෫༴ࡡቌኬ࡞ࡻࡽể⤎ 5 ms 10µV ᬏࡡᠺ㛏㏷ᗐኬࡀࡂ㸡ࡱࡒ DMSO ࡡ⣵⬂හࡡᾈ ㏩㛣▯࠷ࡡ⌦⏜࡚㸡DMSO ࡞ࡻࡾಕ㆜࣒࢜ ࢼࢫ࣑༎ฦⓆᥱ࡚ࡀ࠾ࡖࡒ࡛⩻࠻ࡼࡿࡾ㸣⥾ះ ⤎Ἢ࡞ࡻࡽ⣵⬂ࢅ⤎ࡈࡎࡒሔྙ㸡ể⤎ᬏࡡᠺ㛏 B ㏷ᗐ࠵ࡾ⛤ᗐᢒโࡈࡿࡾࡒࡴ㸡⤎࡞ఔࡖ࡙⏍ࡋ ࡾ⣵⬂හአࡡ⁈ᾦ⃨ᗐࡡን㏷ᗐࡵᢒ࠻ࡼࡿ㸡ࡱࡒ DMSO ࡞ࡻࡾ⭯ࡡಕ㆜ష⏕ᶭ⬗ࡊࡒ࡛⩻࠻ࡼࡿࡾ㸣 ᚃ㸡ể⤎ᬏࡡᠺ㛏࡞ఔ࠹⁈ᾦࡡ⃨ᗐንࡷງᏕⓏ Fig. 3. Spontaneous action potential of cryopreserved neuronal cells (freezing condition ձ) on the multi-array electrode at 16 days after culture. The action potential was measured by the electrode (marked by circle in Fig. 2). A: action potentials were generated at constant rate during 40 seconds. B: Higher magnification of one of these signals. ௧୕ࡡ⤎ᯕࢅࡱ࡛ࡴࡾ࡛㸡Table 2 ࡡࡻ࠹࡞ࡾ㸣 ᙫ㡢࡞ࡗ࠷࡙ふῼࡊ㸡࣒࢜ࢼࢫ࣑ࢅ᪺ࡼ࠾࡞ࡊ࡙࠷ ࡂᚪさ࠵ࡾ㸣 ࡱ ࡛ ࡴ ฦᩋࡊࡒิࣚࢴࢹኬ⬳⓮㈻⏜ᮮ♼⤊⣵⬂ࢅ⤎ ಕᏋࡊ㸡ࡐࡡᶭ⬗ࡡḿᖏᛮࢅన┞ᕣ㢟᚜㙶ふᐳ࠽ࡻ ࡦ㞹ẴⓏὩິࢅ☔ヾࡌࡾࡆ࡛࡚⾔ࡖࡒ㸣ᇰ㣬⏕ᇰᆀ ձࡡ⤎᪁Ἢ࡞ࡻࡽಕᏋࡈࡿࡒิ♼⤊⣵⬂ࡢ㸡よ ࡞⤎ಕ㆜࡛ࡊ࡙ 5㸚 dimethylsulfoxide ࢅຊ࠻㸡 ᚃࡡⓆ㐡≟Ἓᮅ⤎ࡡ⣵⬂࡛㢦జࡊ࡙࠽ࡽ㸡⮤ 㸢3Υ/min㹳㸢150Υ/min ࡡ⠂ᅑ࡚෫༴㏷ᗐࢅንࡈ ⓆⓆℾࡵ☔ヾࡈࡿࡒࡆ࡛࠾ࡼ㸡ᶭ⬗ࢅ⥌ᣚࡊࡒࡱࡱ ࡎࡒ㸣 1 㐄㛣ಕᏋᚃ᛬㏷よࡊ࡙ᇰ㣬ࡊࡒ⤎ᯕ㸡ᮇ ♼⤊⣵⬂Ẓ㍉Ⓩ㧏࠷☔❟࡚⤎ಕᏋ࡚ࡀࡾࡆ࡛ ◂✪ࡡ᮪௲හ࡚ࡢ෫༴㏷ᗐ㐔࠷⸵⏍⋙㧏࠷ ࢂ࠾ࡖࡒ㸣ࡱࡒ㸡5㸚DMSO ࢅ⤎ಕ㆜࡛ࡊ࡙⏕ ࡆ࡛ࢂ࠾ࡖࡒ㸣ࡱࡒᮅ⤎⣵⬂࡛ྜྷ➴ࡡ⮤ⓆⓆℾ ࠷ࡒ㸡⤎㏷ᗐ㐔࠷⸵⏍⋙㧏࠾ࡖࡒ㸣 ☔ヾࡈࡿ㸡ᶭ⬗Ⓩ࡞ࡵಕᏋࡈࡿ࡙࠷ࡾࡆ࡛☔ヾ ࡈࡿࡒ㸣 Table 2. Summary of cryopreserved primary rat cortical neuronal cells in different cooling rates. ㅨ ㎙ ձ㸢ΥPLQ ղ㸢ΥPLQ ճ㸢ΥPLQ մ㸢ΥPLQ Microscopic observations Same as control Same as control Fewer number Not recovered Spontaneous action potential Same as control Fewer number Sometimes Not measured ୌ⯙࡞⤎ಕ㆜ࡡ⥾ះ⤎㸝⣵⬂አ⤎㸞࡞ཀྵ ᮇ◂✪ࡡୌ㒂ࡢ㸡ᾇ㐠ኬᏕ❻⛁Ꮥᢇ⾙භྜྷ◂✪ ࢬࣤࢰ࣭ࡡ㸝ᰬ㸞ࣈ࣏࣭ࣚࣛࢬࣜ♣࡛ࡡභྜྷ◂✪ ࣈࣞࢩ࢘ࢠࢹ㸡࠽ࡻࡦ⛁Ꮥ◂✪㈕ຐ㔘ᴏ㸝ㄚ㢗 ␊ྒ 17340125㸞࡞ࡻࡽ⾔ࢂࡿࡒ㸣 ᩝ ⊡ multimicroelectrode surface. IEEE Trans. Biomed. 1) Წ⃕ୌ㑳㸯ࢂࡿࢂࡿࡢ⏍మ⤄⧂ࡡ⤎ಕᏋ࡞ࡗ ࠷࡙రࢅ▩ࡼ࠷࠾㸣Ὼ⏍∸ᕝᏕఌヽ㸡44㸡 Eng., 26, 273-279 (1979) 11) Marco-Jiménez, F., Garzón, D. L., Peñaranda, D. S., 10-12 (1998) Pérez, L., Viudes-de-Castro, M. P., Vicente, J. S., 2) Marris, E.: Grey matters. Nature, 444, 808-810 Jover, M. and Asturiano, J. F.: Cryopreservation of (2006) European eel spermatozoa: Effect of dilution ratio, 3) Ⓣᶌ㸯㹷ࡱࡵࡡ㹸ࢅ㛏ࡂࡵࡒࡎࡾ̿෫࣬෫ fetal bovine serum supplementation, and ⶮ࣬よᢇ⾙㸣㹷ࣁ࢛࡞Ꮥࡦࣁ࢛ࢅ㉲࠻ࡾ cryoprotectants. Cryobiology, 53, 51-57 (2006) ᕝᏕ࠾ࡼࣁ࢛ࡡࣈ࣭ࣞࢲ㹸㸡ῳ㎰ḿ ⥽㸡 12) Jinbo, Y., Robinson, H. P. 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