Korean Chemical Engineering Research

전체 글

(1)Korean Chem. Eng. Res., Vol. 43, No. 1, February, 2005, pp. 103-109. ëÇ ·<[

(2) % |ð +£ß £ÃÛ£ Lð. oSL <·< ¿ ðÃG <<D û* †. D¬H/ àê¬H @·dHàHê 100-715 ÖQ OC 1 3Í 26 *õ§¬H/ àê¬H dà·@àHz 120-749 ÖQ ¬éC Uô 134 (2004¸ 11ö 4³ [¤, 2005¸ 1ö 13³ >). Effect of Environmental Factors on the Growth of Rabbit Oral Keratinocytes Moon-Young Yoon, Hee-Jung Park, Doo-Hoon Lee, In-Keun Jang, Jung-Keug Park† and Woo-Sik Kim* Department of Chemical and Biochemical Engineering, Dongguk University, 26, 3-ga, Pil-dong, Chung-gu, Seoul 100-715, Korea *School of Chemical Engineering and Biotechnology, Yonsei University, 134, Sinchon-dong, Seodaemun-gu, Seoul 120-749, Korea (Received 4 November 2004; accepted 13 January 2005). ß È. Ù CãYL )§¯ ~- Ú ³ó ör, §¯ ßÑ Âj2 h ¯É Ñ ¬K õCõ T75-ñ¶) ¿õ ÄGñ ¤}G. Ù CãYLÆ)s í(biopsy)K á 6?U(trypsin) Ás-örs «ÄGñ 0.25 cm YL Æ)k³z 1.92±0.59 10 í YL )§¯õ ¡¤O ¤ ÀÉ. ¡¤K YL )§¯õ 50 mg/L BPE(bovine pituitary extract), 5.0 µg/L EGF(human recombinant epidermal growth factor), 0.15 mM Ca s WKK K-SFM(keratinocyte serum free medium)s 10 mL ÄGñ ³ó K ê 8³ OÑ Ä,,

(3) Ñ §¯Í ¯d(confluent)Gj ßG Ê ÍQÑo 2.45³«É. ³ó K §¯õ ¡¤K á (ÜÍ, (z), Íí ÜÍÍ )§¯ ßÑ Âj 2 s Æ G.

(4) Í (2 §¯ ßÑ z_J¯ Áêõ ÷,Ê,

(5) Í ÍWÑ ¶ §¯ ßo Ã ¡dÍ ÇÉ. (z)Í ÍWÑ ¶ §¯ ßo ÝGÊ, Í ÍO¤´ §¯ ßo ÍGk 2.0 mMÑ MJjõ ÷É. « k³ Ù CãYL )§¯õ T75-ñ¶)¿õ ÄGñ G2 Î 50 mg/L BPE, 5.0 µg/L EGF, 2.0 mM Ca s WKK K-SFMs 10 mL ÄG2 ÆQ« Íß JYGÊ ÍQÑo 1.32³« É. «6K õCê2 á YL O 4n¶ )z, ÎL Ù ¯Ñ ÊéG2 )§¯ s .K à_íè«÷ @ íä, ªÑ KÄK _Ýõ fàO ak³ 0ê. 2. 6. 2+. 2+. Abstract - Isolation and primary culture technique of rabbit oral keratinocytes, and the study for effect of environmental factors on the cell growth were carried out in T75-flask. 1.92±0.59×106 viable cells were isolated by trypsin enzymatic digestion method from 0.25 cm2 biopsy of rabbit oral mucosa. Primary culture with 10 mL of K-SFM containing 50 mg/L BPE, 5.0 µg/L EGF and 0.15 mM Ca2+ showed confluence after 8 days and doubling time was 2.54 days. Effect of medium types, medium volume and supplement types on the cell growth was investigated after the cultured keratinocytes had been harvested from primary confluence. Serum addition showed adverse effect and the increase of serum concentration didn’t have an effect on the cell growth. The increase of medium volume decreased the cell growth. The increase of calcium concentration increased the cell growth and 2.0 mM was optimum value. In conclusion, when rabbit oral keratinocytes was cultured in T75-flask, the most effective conditions was to use 10 mL of K-SFM containing 50 mg/L BPE, 5.0 µg/L EGF and 2.0 mM Ca2+, and doubling time was 1.32 days. This study can provide the useful informations to develop a process and design a bioreactor for the culture of keratinocytes in human body like skin and cornea, as well as mucosa. Key words: Keratinocyte, Mucosa, Medium Supplements, Calcium, Environmental Factors, Serum-free Medium. † To. whom correspondence should be addressed. E-mail: [email protected]. ‡. « øéo õ§¬0/ 6ÎR /¤ _¸s ,Ä/ñ ·ÊæÉ5n. 103.

(6) 104. 1.. C «. Oéöá_ö«åöß¯öá_ö6ÎR. gã58

(7) ê ~tÑ gã 5 Üs fMG2 Î p.K gãYL « tDæ

(8) «6K Î )zõ «RG2 a « ³äJ¯ j0ör«[1, 2]. 6÷ «6K )zÆ)s «RO Î )z áÖ gÆQ DDs K(GD AéÑ YLÆ)ê á0 Jk³ kÎ íÊ ,)§¯Q õ§¯Q DÊÄ AéÑ ) z É U8« ¬³ K(ê[3]. 6³ «Rj «Rê ) zÆ)o ¥« É¶M÷ 1« ÿ ¤ ÀD AéÑ hÉÑj2 z JYO ¤ Àk³ YL s j0GD .g2 YL Æ)s «RG2 a« « J«¶ O ¤ À[4, 5]. YLÆ)o hÉ ÉU as ÄG2 a«

(9) ò Mzäs è@Qÿ( :D AéÑ Íß « J«(O, ÉD U Ñ p.K YLÆ)s ¾´ 2 ao M ÍDG³ «Ñ ¬K ¬8k³ á¯ gãÑ

(10) YLÆ)s ¾´ á YL§¯õ ~-Gñ Ñ ß QE 3óòJ¯ ¯àYLÆ)s fÆG2 Æ)àH D¬s ÄG 2 a« Íß àÆ)K ör«¶ O ¤ À[6-9]. K, gãYL )§¯2 )z )§¯Ý ßÍ uíÊ Ñ @ÊD Ñ« LÊ Æ)í(biopsy) áÑ tõ ´L ¤ Àk³ )z j 0õ .K ¯à )Æ) §¯òk³ Ùé « [10]. «6K ¯àYLÆ)s fÆGD .g2 Ñ YL )§¯õ ¬

(11) RQ ¤ À2 h -+Ñ ¬K õgÍ }æ´t G2) 4) «6K õg2 ÂWK =«. )§¯õ GD .Gñ 3T3 KH§¯õ feeder layer³ ÄG2 a« ³äJ¯)[11], « öro Ñ 5 Ü, ÙéJ¯ §¯ í0 Ú ñ K K éfYËs õgG2 ÎÑ ñ6 Í( Hæ2 ¯ÉËs è@Q ¤ À´ «6K feeder layer Äs )GD .K ñ6 Í( ñ )§¯ örË« íèæ´ f. , ¶n(collagen), )³ (fibronectin), ¶Âr(laminin) ço §¯ D0(extracellular matrix) Ñ èê ~ÉËs ,

(12) Ñ ªG2 ör[12], Ä ( õ ¡dQÿ2 ör[13], conditioned medium «÷ complex biologic extractsõ ÄG2 ör[14], D Í( örËs H YGñ ÄG2 ör[15] Ù« Àk÷ «6K örËo ( @í HJ¯ +

(13) Ñ §¯ DDs ÝD .K ör«¶ O ¤ À Ê §¯õ ¡¤K á ÄGD .K IJk³2 «6K örË« W fJ «M÷ kÎ sK örË«. á õgÑ2 T75-ñ¶)¿Ñ Ù gãYL )§¯õ U¾ ßQÿD .Gñ, )§¯ ~- Ú ³ó ê h ¯ÉÑ ¬K s Æ K á ~G. h ÆQs -+GD .Gñ (ÜÍ, ( Ííê (z)Ñ ¬K s Æ GÊ, ( Íío DáJk³ Äæ2

(14) , BPE Q EGF, Ok³ ÆQËs fKG. 2.. /÷ Z tà. ëÇ ·`[

(15) % Ï÷ Z Çs{Ü Ù gãYL )§¯õ ~-G2 QóÑ ¬Gñ Fig. 1Ñ Å. G. Ù2 0ÚÃ ¢+ 4Ù³ 11cê è 2.5 kg å j ¤s ÄGÊ, Ù gã YLÆ)s ®1ÃU( \: 1 ¯W, 1fu(c))k³ £~¾ 4cÊ ]@f(Antibiotic Antimycotic, Invitrogen. Cat.# 600-5240AG)Í 20 µl/mL³ ¯Wê DMEM(Invitrogen Cat.# 12800-017)Ñ ´ -ß5³ ÒäG. 2-1.. ¤@¤ C43× C1 2005 2. + -. ,. Fig. 1. Schematic diagram of procedures to isolate oral keratinocytes from normal rabbit mucosal segment. Mucosal segment, Washing with 70% ethanol and PBS, Incubation for 16 hours at 4 oC in dispase solution, Separation of epidermis and dermis, Chopping and trypsinization, Isolation of 7 epithelial cells, Culture in T75-flask. .. / 1. 0. gã YLÆ)s 70% Ñ!9³ §tGÊ Q Antibiotic Antimycotic 200 µlQ Gentamycin(Invitrogen. Cat.# 600-5710 AD, 0.1 µl/mL) 10 µl Í Ë´À2 10 mL PBS(phosphate buffered saline) ³ 3¡ §tK á 8ê ¤¬Ä Í.³ ,)Q )G(ö@ z.õ £~¾ fMGÊ, 2.4 U/mL l s Í, protease(dispase; Sigma Cat.# P-3417) Ä UÑ Ê 4 CÑ 16QÑ s-K á ¯¹(forceps)s «ÄGñ )@ê ,)@s ~-G. )@s 8êÄ ¤¬ Í.õ «ÄG ñ Â§Gj Éñ á 0.05% T?U(trypsin, Sigma Cat.# T-4799)ê 0.01% EDTAõ WKK PBS ÄUÑ Ê 37 CÑ 10~Ñ s-G Ê 10%(v/v) FBS(fetal bovine serum, Invitrogen Cat.# 12484-028) Í WKê DMEM (Ñ ; 1,000 rpmÑ 5~ ò[~-G. ò[~- á ÙUo `-Ê Q K-SFM(keratinocyte serum free medium, Invitrogen, Cat.# 320-7005PJ) Dá ( 10 mLõ ÍK á )cª(pipetting)s u 5~ YQGñ §¯õ ~-G. @§¯ ª ¤(viable cell count)2 PBSÑ õ¯ 4.0 g/L T?¶ P(trypan blue, Sigma Cat.# T-5526) ÄUk³ +K á hemocytometerõ « ÄGñ ¤}G. ¡¤K §¯2 Ca 0.15 mM, antibiotic antimycotic ]@f õ 10 µl/mL, ( Íí(supplement)³ BPE(bovine pituitary extract, Invitrogen Cat.# 13028-014) 50 mg/LQ EGF(human recombinant epidermal growth factor, Invitrogen Cat.# 10450-013) 5.0 µg/L« WKê K-SFMs ÄGñ GÊ, ( 10 mL s WKK T75-ñ¶)¿(,

(16) J 75 cm )Ñ [ÜGñ 2³ K (õ Ghg c

(17) 37 C³ K(æ2 CO ¯}«Ñ G. 2-2. { Ð£, óKW Ð£, ÇãØ Z { Ë£ Lð Ù gãYL )§¯õ ßQÿ2) (ÜÍ s Æ. GD .Gñ Ë K-SFM Dá (Ñ BPE 50 mg/L, EGF 5.0 µg/L, 1.5 mM Ca õ ÍK Î, Ì MEM (Ñ FBS 10%(v/v) Í K Î, Í DMEM (Ñ FBS 10%õ ÍK Î, Î Ham’s F-12 (Ñ FBS 10% ÍK Îõ WGGñ ÝI. (Íí Ü ÍÑ ¬K s Æ GD .Gñ Ë K-SFM Dá (Ñ K Î, Ì K-SFM Dá (Ñ Íí³ 1.5 mM Ca ê FBS 10%õ ÍK Î, Í K-SFM Dá (Ñ Íí³ 50 mg/L BPE, 5.0 µg/L EGF, 1.5 mM Ca õ ÍK Î, Î K-SFM Dá (Ñ Íí o. o. 2+. 2. o. 2. 2+. 2+. 2+.

(18) CãYL )§¯ ßÑ Âj h ¯É . ³ 50 mg/L BPE, 5.0 µg/L EGF, 1.5 mM Ca ê FBS 10%õ Í K ÎõWGGñ ÝI.

(19) Í sÆ GD .Gñ K-SFM Dá (Ñ BPE, EGF, Ca s ( :Ê FBS 0, 5, 10, 15, 20%. ³ Í Gñ Ù gãYL )§¯ ßs WGG. s Æ GD .Gñ 50 mg/L BPE Q 5.0 µg/L EGF õ WKK K-SFM (Ñ Ca õ 0.1, 0.5, 1.0, 1.5, 2.0, 2.5 mM« æ´ ÆQK á §¯ ßs WGG. D HÇ Y×Ñ (z)2 10 mL³ G. (z)Ñ ¬K o T75-ñ¶)¿ Ñ 50 mg/L BPE, 5.0 µg/L EGF, 1.5 mM Ca WKK K-SFM (õ ÎÎ 10, 15, 20 mL æ´ ÆQGñ K á §¯ ßs WGG. HÇ Î ÆQo ³ó á ¡¤K §¯õ cm ¢ 3.5 10 í ³ ÎÎ (õ WKGÊ À2 T75-ñ¶)¿ Ñ [ÜGñ 37 C³ K(æ2 CO ¯}«Ñ 4³ K á hemocytometerõ «ÄGñ @§¯ ª¤(viable cell count)õ ¤}G Ê, (G cD2 2³³ G. 2+. 2+. 2+. 2+. 2. 3. o. 2. 3.. ûG Z +{. ëÇ ·`[

(20) % Ï÷ Z Çs {Ü Ù³z gãYLÆ)s ¾´ á YL )@Os ~-Gñ T?Uk³ Ás- K á õJ¯ )cª(pipetting)k³ 0.25 cm YL Æ)k³z 1.92±0.59 10 í YL )§¯õ ¡¤O ¤ ÀÉ. ¡¤ê §¯õ 50 mg/L BPE, 5.0 µg/L EGF, 0.15 mM Ca s WKK K-SFMs ÄGñ 10 mL 2³K (õ Ghgc

(21) K ê 8³ OÑ ,

(22) Ñ ¯d(confluent)æj ßG . Fig. 22 ³ó ê_Ñ QÑÑ !¶ §¯ ßs Ý D .Gñ HÂ GÑ Öo ,s ÷ a«. ð DÑ2 ß« uíj «P´(( :I(O, 4³ áz2 u ñ ³ ßG. U0 á §¯¤õ +_K ê ðD [Ü

(23) « 2.6 10 í §¯¤³z 8³ 8 (2.5±0.3) 10 í §¯¤³ RGñ ðDÑ Wg u 9.6 ßs ÷É 3-1.. 2. 6. 2+. 5. 6. Fig. 2. Light microscopic pictures for the primary culture of normal rabbit oral keratinocytes at Day 1 (A), Day 2 (B), Day 4 (C) and Day 8 (D). After the keratinocyte was isolated from mucosal segment, 3.5×103/cm2 of cells were inoculated into T75-flask with 10 mL of K-SFM containing 50 mg/L BPE, 5.0 µg/L EGF and 0.15 mM Ca2+. Photographs at a magnification of ×100 were taken with a camera attached to a phase-contrast microscope. Bar indicates 200 µm.. 105. Ê, ÍQÑ(doubling time)o 2.45³«. Daniels Ù[16] ÝÊ Ñ G

(24) hÑ é ÄGÊ À2 )§¯ örÑ ¬K Æ Ñ 3T3 KH§¯õ feeder layer³ ÄGñ

(25) « WKê Ê (õ ÄG2 ör« 53%³ ÷

(26) Ê, I å

(27) (õ ÄG2 ör« 43%³ ÷

(28) .

(29) s W KK (õ ÄG2 Î2

(30) 1òk³ ¯K ÇÍéf Ù ñ6 ÞÛK éfõ è@Q ¤ Àk³ é å

(31) ( õ ÄGs2 ök³ MhGÊ À. I s WKK å

(32) ( Yo feeder layerõ ÄG2 ÎÝ §¯ Ñ YÒG2 ÎÍ QÊ §¯ ßÍ .s ³ó DÑ« 2c _ U12 a«. 6÷ á õgÑ2 ³ó DÑ« 8³³ ÷

(33) 2) « ê2 (ÜÍ, Íí ÜÍ, Ñ ÄD ,

(34) J ÍQ (z) ÝQ ço h ¯ÉÑ K k³ 0æÊ, «: ¯ÉË« §¯ autocrine èn Ñ K «¯ õ ÍQE §¯ ßs

(35) Ï ñ,QZD Aék³ 0ê. «: h ¯ÉË Ñ ¬g Ö

(36) gJ k³ Æ GD .Gñ ³ó K §¯õ ¡¤Gñ h ¯É Ñ ¬K ÍJ¯ Y×s ª ,}G. 3-2. { Ð£, óKW Ð£, 5ø, ÇãØ Z { Ë£ Lð Ù gãYL )§¯õ ßQÿ2) å

(37) Dá (¯ K-SFMÑ Íí³ 50 mg/L BPE, 5.0 µg/L EGF, 1.5 mM Ca s WKK (Q

(38) « WKê Í( ( ÜÍ s Æ G . Fig. 3Ñ ÷ asF K-SFMÑ Íís WKK ÎÍ MEM, DMEM, F-12 (Ñ

(39) s ÍK ÎÝ 37.5%, 106.3%, 112.9%³ §¯ ß« ÍG. « ê³z K-SFM Dá (Ñ BPE, EGF, -Ê

(40) ÍÍ §¯ ßÑ ´¥ s Âj2( <4ÝD .K Y×s G . K-SFM Dá (Ñ K ÎÑ Wg K-SFM Dá ( Ñ 50 mg/L BPE, 5.0 µg/L EGF, 1.5 mM Ca s ÍK ÎÍ 2+. 2+. Fig. 3. Effect of media types on the growth of rabbit oral keratinocytes. After the cultured keratinocyte had been harvested from primary confluence, 3.5×103/cm2 of cells were inoculated into T75-flask with 10 mL of each media. Supplements were used at the concentrations of 50 mg/L BPE, 5.0 µg/L EGF, 1.5 mM Ca2+ and 10% (v/v) FBS. Cell number was counted after 4 days of culture. Korean Chem. Eng. Res., Vol. 43, No. 1, February, 2005.

(41) Oéöá_ö«åöß¯öá_ö6ÎR. 106. Fig. 4. Effect of supplements on the growth of rabbit oral keratinocytes. After the cultured keratinocyte had been harvested from primary confluence, 3.5×103/cm2 of cells were inoculated into T75-flask with 10 mL of K-SFM containing different supplements. Supplements was used at the concentration of 50 mg/L BPE, 5.0 µg/L EGF, 1.5 mM Ca2+ and 10% (v/v) FBS. Cell number was counted after 4 days of culture.. §¯ ß« Í GÊ, 50 mg/L BPE, 5.0 µg/L EGF, 1.5 mM Ca s ÍK K-SFMÑ

(42) s

(43) ÍK Î2 §¯ ß« 1 ¾s 33.3% ÝG. K-SFM Dá (Ñ Íí³ BPEQ EGF õ fGÊ 1.5 mM Ca ê 10%(v/v)

(44) Os ÍK Î2 BPE, EGF, s WKK (Ñ Wg §¯ ß« 68.2% ÝG. « ê³z BPE, EGF, « §¯ ßÑ _« îñG2 ak³ ¶æÊ

(45) Í2 z_J¯ Áêõ ÷2 ak³ 0ê. Wang Ù[17]o BPE2

(46) sF 0ê ß¯ÉË ÞY« Ê, )§¯ ßs ñ,G2 òOs G(O ,)§¯¯ KH§ ¯ ßs ñ,G(2 :Ê, )§¯ ~dõ IgG2 ak³ ÝÊGk÷ 4) «aÑ ¬K _eK ÊÄ èn o é(( :Ê À. K, BPE 4Âô Æ o bovine pancreatic trypsin inhibitor(bPTI)sF T?U s-Ñ g )§¯õ ¡¤K á 4 À2 T?U l s IgO ¤ ÀÊ BPEÑ2 EGF ~« WK æ´ À( :o ak³ ÝÊG. Kamata Ù[18]o BPEÍ Í æ( :o å

(47) ( Î )§¯Í ßG( :IÊ, BPE õ WKG( :2 å

(48) (Ñ EGF, KGF, IGF-zê bFGF ç o ß¯ÉË ÍÍ )§¯ ßs ¿j ÉKÊ ÝÊ G. 6÷ Cook Ù[19]o Æ foreskin )§¯ Î § ¯ Í cm ¢ 5 10 í « ³ A peptide growth factorÍ Ç´ )§¯Í õJk³ ßGÊ, IGF, EGF, TGF-α, FGFõ Í³ ÍK Î ßõ ¿j àü( :IÊ ÝÊG. «: ñ ê2 )§¯Í ßO A autocrine èn Ñ K A é«¶ O ¤ ÀÊ, U¾ EGF-R(EGF-receptor)Q a -ÑÃ (ligands)Í autocrine[20, 21]ê paracrine ßÆQ[22-24]Ñ y®K òOs G2 ak³ <s À. å

(49) (õ ÄG2 Î , )§¯Í ~WG2 «¯ òOs G2 EGFõ ¯WG2 ß¯ÉËê BPE ço 0Ës Íg c´t G2 a« ³ä 271.8% 2+. 2+. 2. 3. ¤@¤ C43× C1 2005 2. Fig. 5. Effect of serum concentrations on the growth of normal rabbit oral keratinocytes. After the cultured keratinocyte had been harvested from primary confluence, 3.5×103/cm2 of cells were inoculated into T75-flask with 10 mL of K-SFM containing different serum concentrations. Supplements was used at the concentration of 50 mg/L BPE, 5.0 µg/L EGF and 1.5 mM Ca2+. Cell number was counted after 4 days of culture.. J«(O á õgÑ2 «: )§¯ autocrine èn s Ê sGñ DáJ¯ Íí³ Äæ2 BPE, EGF, Os ¬ k³ G. D Y×Ñ

(50) ÍÍ §¯ ßÑ z_J¯ s ÷ É2),

(51) Ñ ¡dõ cÉs A §¯ ßs IgG2(õ < 4ÝD .Gñ

(52) Ñ ¡dõ c

(53) §¯ ßs Æ Gñ ÝI . Fig. 5Ñ ÷ asF

(54) Í ÍWÑ !¶ §¯ ßo Ã ¡dõ ÷( :IÊ 50 mg/L BPE, 5.0 µg/L EGF, 1.5 mM Ca s WKK K-SFMÑ Wg 68.2% ÝG. 6³

(55) Í Ok³2 )§¯ ßs òO¾ K(GDÍ ´sÒ ak³ ¶ ê. ³äJk³ ,)§¯ ÎÑ2 (Ñ

(56) s ÍGñ Gñt G(O )§¯ Î2

(57) « 1¾s ßs Ig G. «: ê2 ¯Æ) gÆ ,)§¯ Î

(58) î« Ë ´Q À(O )§¯b(

(59) î« ÊéG( :Ê ,)§¯Í ß G

(60) ~WK «¯Ñ g )§¯Í ßñ,s ³kÿD AéÑ

(61) o )§¯Ñ z_J¯ Áêõ ÷2 ak³ 0 ê. «6K «K³ )§¯õ O A

(62) s WKK (Ñ ,)§¯òOs G2 3T3 KH§¯õ «ÄGñ feeder layerõ OÇ á .Ñ )§¯õ G2 D¬s ÄG2)[11], « öro ß« µfê 3T3 §¯õ ] gWG2 a« Ä«G( : Ê, 3T3 §¯ @Ê(viability)Í feeder³ J¢G( :s ¤ Àk,

(63) ~Ë« KH§¯õ êGj ßQ ¤ Às O 4n¶,

(64) 0« 8_K 0s ÷D AéÑ )§¯ ß 8Ñ éfõ è@Q ¤ À.

(65) o ³äJ k³ í§¯ Ñ ßs (òG2 òOs G2), DíL, ß¯É, Y0(binding proteins)ço Qo ~Ës fàG (O áº-4, K« ñ¶ K(mycoplasma), à«6) 1 ço ÙéJ¯ .×®¯s fàO ¤ À.

(66) o K k ¡~(batch) ¥³ ¡dÍ [GÊ, ¯Æ)s O A ÄG2 Î2

(67) ò 2+.

(68) CãYL )§¯ ßÑ Âj h ¯É . 107. Fig. 6. Effect of medium volume on the growth of normal rabbit oral keratinocytes. After the cultured keratinocyte had been harvested from primary confluence, 3.5×103/cm2 of cells were inoculated into T75-flask with K-SFM containing 50 mg/L BPE, 5.0 µg/L EGF, 1.5mM Ca2+ of different medium volume. Cell number was counted after 4 days of culture.. Fig. 7. Effect of Ca2+ concentrations on the growth of normal rabbit oral keratinocytes. After the cultured keratinocyte had been harvested from primary confluence, 3.5×103/cm2 of cells were inoculated into T75-flask with 10 mL of K-SFM containing 50 mg/L BPE, 5.0 µg/L EGF and different Ca2+ concentrations. Cell number was counted after 4 days of culture.. Mzä Ùs ÷ ¤ À2 Y« À. «6K «K AéÑ é §¯ s .Gñ å

(69) (õ «ÄGs2 ök³ Mh GÊ À. Fig. 6o ³K ,

(70) J ÆQÑ (z)Í )§¯ ß Ñ Âj2 s ÷ Öé¯), (z)Í 10 mL, 15 mL, 20 mL ÍWÑ !¶ )§¯ ß« ÝG2 êõ ÷ É. «ao (z)Í ÍWÑ !¶ §¯Í ~WG2 « ¯ õ ÝQÿD Aék³ 0ê. 6³ )§¯õ UGj RQÿ2 .g2 (z)õ kñ ÄG2 a« §¯ ßõ ÍQÿ2) K-O ak³ ¶ê. )§¯ ßÑ Âj2 s Æ Gñ Fig. 7Ñ ÷. É. Í ÍO¤´ §¯ ß« ÍGk 2.0 mM. Ñ MJjõ ÷É. Fig. 8o ÎÎ Ñ õ J¯ ª¬ s G

(71) passge number 2Ñ Â ,s ÷ a¯) §¯Ho Í ÍO¤´ YY è³Gj ¡dG2 s ÷É. « ê³ 0.1 mM I Ý 2.0 mM Ê s WKK K-SFMÑ §¯ ß« 76.9% ÍGÊ ÍQÑo 1.32³«É. ä

(72) Í ÍO¤´ §¯H« ¡G2 s e¯O ¤ ÀÉ. Barbara Ù [25]o I s WKK å

(73) (Ñ )§¯Í ßK Î §¯ ª

(74) « ÿÅÊ kÎ oQs Ý«2) «ao §¯Q §¯Ñ zôDt ; Aék³ GÊ, Í Í. Fig. 8. Morphology of normal rabbit oral keratinocytes at passage number 2 cultured in T75-flask with 10 mL of K-SFM containing 50 mg/L BPE, 5 µg/L EGF and 0.1 mM Ca2+(a), 0.5 mM Ca2+(b), 1.0 mM Ca2+(c), 1.5 mM Ca2+(d), 2.0 mM Ca2+(e), and 2.5 mM Ca2+(f). Photographs at a magnification of ×100 were taken with a camera attached to a phase-contrast microscope at Day 4 after inoculation. Bar indicates 200 µm. Korean Chem. Eng. Res., Vol. 43, No. 1, February, 2005.

(75) Oéöá_ö«åöß¯öá_ö6ÎR. 108. O¤´ §¯Q §¯Ñ õ« ÆÊg(2 Hs ÷É2) «ao Í Ãä1(cadherin)s ¯WG2 §¯ zô ~ É(intracellular adhesion molecules) DDs l dGD Aék³ ÝÊG. Ê Ñ §¯Q §¯Ñ õo P-Ãä1 ê E-Ãä1 JÑ K ak³ ½æÉÊ[26], «: Ãä 1 l d2 §¯Q §¯Ñ õ O 4n¶ §¯ y@d (stratification)Ñ îñG2 ak³ <s À[27]. «6K §¯H s G2 ao §¯Í ~dG

(76) Æ)ê ço gÆõ OËD .K ª³ 0ê. Fig. 8Ñ ÷ Â ,Ñ ¥³ §¯ Hs WGGñ Ý

(77) «6K ÝÊQ K K as < ¤ À. K, Í §¯ ßÑ Âj2 Ñ ¬g2 Tsao Ù[14]o õ 1.0 mM WKGÊ À2 (Ñ )§¯õ K Î §¯ ß« I¾ ÝG2 ak³ ÝÊG. Kamata Ù[18] )[ Æfg OÇ å 0 Y ((protein-free synthetic medium)Ñ 1.27 mM³ õ ÆQK á Æ gãYL )§¯õ K ê ß« M «P´(( :IÊ H& UM¾ ~dæÉÊ ÝÊG. 6÷ BoyceQ Ham[28] ÝÊÑ G

(78) Í ÍO¤´ )§¯ ß« ÍG Ê 0.3 mMÑ MJjõ ÷É(O 1.0 mM Ê Ñ BPEõ WKK å

(79) ( ÎÍ BPEõ WKG( :o ÎÝ §¯ ß« 900% « j ÷

(80) . U¾ 1.0 mM Ê Ñ §¯~dõ ÷2 ¯É¯ cornified envelopes õ ÷2 §¯Í BPEõ WKG( :o ( Î 20%õ Ý ¯ ä

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(86) Ï ñ, æ2 ak³ 0ê. 4.. û «. Ù gãYLÆ)k³z )²f(dispase) ÁQ T?U Á~görs «ÄGñ YL )§¯õ ~-K ê YLÆ)k³z 1.92±0.59 10 í @§¯(viable cell) õ ¡¤O ¤ ÀÉ. ¡¤K YL )§¯õ 2.6 10 í [ÜGñ 50 mg/L BPE, 5.0 µg/L EGF, 0.15 mM Ca K-SFMs 10 mL W KK T75-ñ¶)¿Ñ ³ó K ê 8³ OÑ ÄDÑ § ¯Í ¯d(confluent)æj ßGÊ, §¯¤2 2.5±0.3 10 í³ 9.6 ßGk ÍQÑo 2.45³«É. ³ó K §¯õ ¡¤K á T75-ñ¶)¿õ ÄGñ å

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(96) JÑ (z)Í ÍWÑ !¶ §¯ ßo ÝGÊ (z)õ 10 mL ÄK ÎÑ Wg 15 mL ÄK Î 9%, 20 mL ÄK Î 30%b( )§¯ ß« ÝG. «ao (z)Í ÍO¤´ §¯Ñ ~Wæ2 «¯. Í ÝGD Aék³ 0ê. Í ÍO¤´ §¯ ßo ÍGk 2.0 mM Ñ M¬jõ ÷É. 0.1 mM I Ý 2.0 mM Ê s WKK K-SFMÑ §¯ ß« 76.9% ÍG. « k³ Ù gãYL )§ ¯õ T75-ñ¶)¿õ «ÄGñ G2 Î 50 mg/L BPE, 5.0 µg/L EGF, 2.0 mM Ca s WKK K-SFMs 10 mL ÄG2 h Æ Q« Íß JYGÊ, ÍQÑo 1.32³«É. «6K õgê 2 á YL O 4n¶ )z, ÎL Ù ¯Ñ ÊéG2 )§¯ s .K à_íè«÷ @íäD ªÑ KÄK _Ýõ fà O ak³ 0ê. EGF, 1.5 mM Ca2+. 2+. 2+. 2+. 2+. 2+. [ ÷ á õg2 ÝQÞ(z 0àH aYD¬ íè Æ(êfdD 00(òÑ g ¤}æÉk «Ñ Ý Ã?n.. PJ1-PG1-CH12-0006). +S 1. Teichgraeber, J., Larson, D. and Castaneda, O., “Skin Grafts in Intraoral Reconstruction,” Arch. Otolaryngol., 110, 528-532(1984). 2. Schramm, V. L. and Myers, E. N., “Skin grafts in Oral Cavity Reconstruction,” Arch. Otolaryngol., 106, 528-532(1980). 3. Ueda, M., Ebata, K. and Kaneda, T., “In Vitro Fabrication of Bioartificial Mucosa for Reconstruction of oral Mucosa: Basic Research and Clinical Application,” Ann. Plast. Surg., 27(6), 540-549(1991). 4. Chase, D. C., Gattozzi, J. G. and Smith, K., “Palatal Graft in Preprosthetic Surgery,” Oral. Surg., 45, 516-527(1978). 5. Fry, T. L. and Woods, C. I., “Readily Available Full-Thickness Mucous Membrane Grafts,” Arch. Otolaryngol. Head Neck Surg., 113, 770-771(1987). 6. Hata, K., Kagami, H., Ueda, M., Torii, S. and Matsuyama M., “The Characteristics of Cultured Mucosal Cell Sheet as a Material for Grafting: Comparison with Cultured Epidermal Cell Sheet,” Ann. Plast. Surg., 34, 530-538(1995). 7. Izumi, K., Feinberg, S. E., Iida, A. and Yoshizawa, M., “Intraoral Grafting of an ex vivo Produced Oral Mucosa Equivalent: a Pre-.

(97) CãYL )§¯ ßÑ Âj h ¯É liminary Report,” Int. J. Oral Maxillofac. Surg., 32, 188-197(2003). 8. Moriyama, T., Asahina, I., Ishii, M., Oda, M., Ishii, Y. and Enomoto, S., “Development of Composite Cultured Oral Mucosa Utilizing Collagen Sponge Matrix and Contracted Collagen Gel: a Preliminary Study for Clinical Applications,” Tissue Engineering, 7(4), 415-427(2001). 9. Fujiyama, C., Masaki, J. and Sugihara, H., “Reconstruction of the Urinary Bladder Mucosa in Three Dimensional Collagen Gel Culture: Fibroblast-Extracellular Matrix Interactions on the Differentiation of Transitional Epithelial Cells,” J. Urol., 153, 20602067(1995). 10. Ueda, M., Hata, K., Horie, K. and Torii, S. “The Potential of Oral Mucosal Cells for Cultured Epithelium: a Preliminary Report,” Ann. Plast. Surg., 35(5), 498-504(1995). 11. Rheinwald, J. C. and Green, H., “Serial Cultivation of Strains of Human Epidermal Keratinocytes: Formation of Keratinocyte Colonies From Single Cells,” Cell, 6, 331-343(1975). 12. Liu, S. C. and Karasek, M., “Isolation and Growth of Adult Human Epidermal Keratinocytes in Cell Culture,” J. Invest. Dermatol., 71, 157-162(1978). 13. Hennings, H., Michael, D., Cheng, C., Steinert, P., Holbrook, K. and Yuspa, S. H., “Calcium Regulation of Growth and Differentiation of Mouse Epidermal Cells in Culture,” Cell, 19, 245-254(1980). 14. Tsao, M. C., Walthall, B. J. and Ham, R. G., “Clonal Growth of Normal Epidermal Keratinocytes in Defined Medium,” J. Cell. Physiol., 110, 219-229(1982). 15. Hawley-Nelson, P., Sullivan, J. E., Kung, M., Hennings, H. and Yuspa, S. H., “Optimized Conditions for the Growth of Human Epidermal Cells in Culture,” J. Invest. Dermatol., 75, 176-182(1980). 16. Daniels, J. T., Kearney, J. N. and Ingham, E., “Human Keratinocyte Isolation and Cell Culture: a Survey of Current Practices in the UK,” Burns, 22, 35-39(1996). 17. Wang, H. J., Chen, T. M., Cheng, L. F., Cheng, T. Y. and Tung, Y. M., “Human Keratinocyte Culture using Porcine Pituitary Extract in Serum-Free Medium,” Burns, 21(7), 503-506(1995). 18. Kamata, N., Yokoyama, K., Fujimoto, R., Ueda, U., Hayashi, E., Nakanishi, H. and Nagayama, M., “Growth of Normal Oral Keratinocytes and Squamous Cell Carcinoma Cells in a Novel Protein-Free Defined Medium,” In Vitro Cell. Dev. Biol.-Animal, 35, 635-641(1999). 19. Cook, P. W., Pittelkow, M. R. and Shipley, G. D., “Growth Fac-. 109. tor-Independent Proliferation of Normal Human Keratinocytes: Production of Autocrine- and Paracrine-Acting Mitogenic Factors,” J. Cell. Physiol., 146, 277-289(1991). 20. Piepkorn, M., Lo, C. and Plowman, G., “Amphiregulin-Dependent Proliferation of Cultured Human Keratinocytes: Autocrine Growth, the Effects of Exogeneous Recombinant Cytokine, and Apparent Requirments for Heparin-Like Glycosaminoglycans,” J. Cell Physiol., 159, 114-120(1994). 21. Pittelkow, M. R., Cook, P. W., Shipley, G. D., Derynck, R. and Coffey, R. J., “Autonomous Growth of Human Keratinocytes Requires Epidermal Growth Factor Receptor Occupancy,” Cell Growth Differ., 4, 513-521(1993). 22. Dulgosz, A. A., Cheng, C., Dening, M. F., Dempsey, P. J., Coffey, R. J. and Yuspa, S. H., “Keratinocytes Growth Factor Receptor Ligands Induce Transforming Growth Factor Alpha Expression and Activate the Epidermal Growth Factor Receptor Signaling Pathway in Cultured Epidermal Keratinocytes,” Cell Growth Differ., 5, 1283-1292(1994). 23. Ristow, H. J., “Studies on Stimulation of DNA Synthesis with Epidermal Growth Factor and Insulin-Like Growth Factor-I in Cultured Human Keratinocytes,” Growth Regul., 6, 96-109(1996). 24. Vardy, D. A., Kari, C., Lazarus, G. S. and Jensen, P. J., Zilberstein, A., Plowman, G. D. and Rodeck, U., “Induction of Autocrine Epidermal Growth Factor Recepter Ligands in Human Keratinocytes by Insulin/Insulin-like Growth Factor-I,” J. Cell. Physiol., 163, 257-265(1995). 25. Marsh, B. C. R., Massaro-Giordano, M., Marshall, C. M., Lavker, R. M. and Jensen, P. J., “Initiation and Characterization of Keratinocyte Cultures from Biopsies of Normal Human Conjunctiva,” Exp. Eye Res., 74, 61-69(2002). 26. Wheelock, M. J. and Jensen, P. J., “Regulation of Keratinocyte Intercellular Junction Organization and Epidermal Morphogenesis by E-Cadherin,” J. Cell. Biol., 117, 415-425(1992). 27. O’Keefe, E. J., Briggaman, R. A. and Herman, B., “Calcium Induced Assembly of Adherens Junctions in Keratinocytes,” J. Cell. Biol., 105, 807-817(1987). 28. Boyce, S. T. and Ham, R. G., “Calcium-Regulated Differentiation of Normal Human Epidermal Keratinocytes in Chemically Defined Clonal Culture and Serum-Free Serial Culture,” J. Invest. Dermatol., 81, 33s-40s(1983).. Korean Chem. Eng. Res., Vol. 43, No. 1, February, 2005.

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