Desarrollo de dispositivo microfluídico para la visualización del fenómeno de la extravasación leucocitaria

ESCUELA TÉCNICA SUPERIOR DE INGENIERÍA INFORMÁTICA G ado en Ingenie ía de la Salud Mención en Ingenie ía Biomédica Desa ollo de disposi i o mic o luídico pa a la isualización del enómeno de la ex a asación leucoci a ia De elopmen o low ocusing de ice o he isualiza ion o leukocy e olling adhesion Realizado po Pablo Mo ales Na a e e Tu o izado po José An onio And ades Gómez* Co- u o izado po Joaquín O ega Casano a** Depa amen o * Biología Celula , Gené ica y Fisiología ** Ingenie ía Mecánica, Té mica y de Fluidos UNIVERSIDAD DE MÁLAGA MÁLAGA, Junio 2018 Fecha de ensa: El Sec e a io del T ibunal Resumen La mic o luídica es un á ea de la mic o ecnología basada en chips de PDMS que es á siendo u ilizada cada ez más en mul i ud de aplicaciones. Una de es as aplicaciones es la in es igación biomédica. La mic o luídica o “Lab on a Chip” se ha con e ido en una mane a de ealiza expe imen os biomédicos y diagnós icos de una mane a ba a a, ápida y e icaz. Cuando se ealizan es udios sob e la ex a asación leucoci a ia u ilizando chips mic o luídicos, podemos obse a la inconsis encia en la ayec o ia de odadu a de los leucoci os debido a un lujo lamina . En es e abajo de in de g ado p esen amos un mé odo pa a cen a la in e az de células en el cen o de canal mic o luídico. Cuando las células ci culan po los sis emas mic o luídicos, las células ienden a ci cula de mane a alea o ia po los canales. Po an o, con el sis ema p opues o en es e abajo, dichas células se án edi igidas a la po ción cen al del canal con el in de ec ea el enómeno de odadu a p esen e en nues o sis ema ci cula o io y así ob ene in o mación más de allada. Los esul ados de es e abajo mues an la u ilidad y la e sa ilidad de es e disposi i o pa a expe imen os elacionados. Palab as cla e: Mic o luídica, Lab on a Chip, Poin o Ca e, Ex a asación Leucoci a ia, Li og a ía blanda, Inmunología, Modelado Mul i ísico, Mé odo de elemen os ini os. Abs ac Mic o luidics is an a ea o echnology based on PDMS chips ha is being inc easingly used o many applica ions. One o said applica ions is biomedical esea ch. Mic o luidics o Lab on a chip poses as a g ea way o cu ing cos s, ime, space and o e all imp o ing e iciency in medical diagnosis and biomedical expe imen s. When s udying he olling cell adhesion beha iou o leukocy es on mic o luidic de ices, we can obse e he inconsis ency in he manne cells oll due o lamina low. In his inal yea disse a ion we p esen a me hod o ocus he low o leukocy es on a ec angula mic o luidic channel o olling cell adhesion assays. When lowing in mic o luidic channels, cells end o ci cula e in a andom manne , he e o e wi h he sys em p oposed, said cells will ci cula e in he cen al a ea o he channel, in o de o op imally ec ea e he olling cell adhesion phenomenon p esen in ou ci cula o y sys em and achie e p ecise in o ma ion ega ding his cellula beha iou . The esul s o his wo k show he iabili y o he sys em and he e sa ili y i may ha e wi h o he ela ed expe imen s. Keywo ds: Mic o luidics, Lab on a Chip, Poin o Ca e, Rolling Cell Adhesion, Hyd odynamic low ocusing, So Li hog aphy, Immunology, Mul iphysics Modelling, Fini e Elemen Me hod. Acknowledgemen s To my pa en s o making he ip o Japan, whe e his disse a ion was de eloped, possible, o ne e losing ai h in me and o always suppo ing my decisions. To my sis e , o encou aging me o y my ha des in e e y s ep along he way. To he P o esso s José An onio And ades Gómez and Joaquín O ega Casano a, o ge ing in ol ed in his p ojec and o hei help de eloping he con en o his disse a ion. To Masahi o Mo osuke, o allowing me o wo k a his lab, whe e I lea n so much. To Sayaka Nomu a and Takuya Ichikawa, o being g ea colleagues and helping me wi h his esea ch. Con en s Chap e 1: In oduc ion ..................................................................................... 13 1.1 Backg ound and Mo i a ion ........................................................................ 13 1.2 Objec i es ................................................................................................... 14 1.3 Con en Ou line .......................................................................................... 15 Chap e 2: The Immune Sys em ....................................................................... 17 2.1 The Immune Sys em .................................................................................. 17 2.1.1 Haema opoiesis ................................................................................... 18 2.1.2 Immune Sys em O gans ...................................................................... 20 2.2 Leukocy e Ac i a ion and Mig a ion ............................................................ 22 2.2.1 Cell Adhesion Molecules ...................................................................... 22 2.2.2 Leukocy e Ex a asa ion ...................................................................... 24 2.2.3 Leukocy e Adhesion De iciency ........................................................... 25 Chap e 3: Mic o luidics .................................................................................... 27 3.1 Wha is Mic o luidics? ................................................................................. 27 3.2 P ope ies o Fluidics .................................................................................. 28 3.3 Shea S ess ............................................................................................... 29 3.4 Types o Fluids ........................................................................................... 29 3.5 Types o Flows ........................................................................................... 30 3.5.1 Lamina low ........................................................................................ 30 3.6 Hyd odynamic Focusing ............................................................................. 32 3.6.1 Conse a ion o Ene gy ....................................................................... 33 3.7 Lab on a Chip ............................................................................................. 34 3.7.1 O gan on a chip ................................................................................... 35 17 Chap e 2: The Immune Sys em 2.1 The Immune Sys em Like all mammals, humans possess a specialized sys em des ined o he p o ec ion and he de ence o ou body; we e e o his mechanism as he Immune Sys em. This complex is composed by e y specialized cells ha unc ion inside e y well o ganized ana omical s uc u es. The e o e o unde s and he impo ance o hese specialized cells we mus s udy hem in conjunc ion wi h he issues and o gans in which hese cells in e ac wi h. I we go back in his o y, e olu iona y speaking, ou immune sys em was de eloped in o de o comba he in ec ions caused by i uses, bac e ia, p o ozoa, ungus and helmin hs. These pa hogens can be esponsible o many in ec ions bo h in acellula and ex acellula , o which he esponse mus be di e en . The immune sys em has de eloped a a ie y o app op ia e de ence mechanisms in o de o igh o each o he di e en pa hogens ha in ec us. In o de o e adica e a pa hogen ha has in ec ed i s hos , he immune sys em mus i s de ec i s p esence and hen ake he necessa y s eps o des oy i . Fo he des uc ion o pa hogens, he human body has de eloped di e se immune mechanisms which a e bo h inna e and adap i e; he di e ence es s in he ac ha hey use di e en me hods o de ec h ea s and in ec ions. In o de o he inna e esponse o wo k, a se ies o molecula pa e ns ha a e p esen in same g oup pa hogens mus be de ec ed. All membe s o a same animal species a e bo n wi h an inna e and immedia e capaci y o de ec ing and des oying nume ous pa hogens which we ha e no been in con ac be o e. The inna e immune esponse is e y e ec i e; howe e , i canno p o ec us om all h ea s. The majo i y o pa hogenic agen s a e single celled o ganisms ha di ide e y quickly; he e o e, hey e ol e a highe a es and e ade he body’s inna e esponse. In o de o a oid his, e eb a es ha e de eloped a pa hogen econnaissance s a egy which enables he de ec ion o o eign agen s ha ha e ne e been encoun e ed be o e. This de ence mechanism is e e ed o as 18 he adap i e immune sys em. Lymphocy es a e he cells which will be esponsible o his adap i e immuni y. Immune cells a e he key playe s in ou immune sys em, enabling de ec ion and des uc ion o o eign pa hogens. The e a e many ypes o cells, each wi h di e en unc ions and cha ac e is ics. Howe e , hei o igin is simila . Immune cells, commonly e e ed o as whi e blood cells o leukocy es, o igina e om he bone ma ow, a semi-solid issue ound in he abecula bone issue. In his myeloid issue, leukocy e p ecu so s a e c ea ed. These “pa en ” cells will hen di e en ia e h ough a p ocess called haema opoiesis o c ea e whi e blood cells. The e a e many ypes o cells in ou immune sys em as seen in Figu e 2.1, all wi h di e en cha ac e is ics bu same objec i e: o des oy o eign pa hogens. Figu e 2.1: Leukocy e de elopmen , om p ogeni o o ma u e di e en ia ed cells (Mikael Häggs öm 2009). 2.1.1 Haema opoiesis All blood cells a ise om a ype o cell called he hema opoie ic s em cell (HSC). S em cells a e undi e en ia ed cells which ha e he unique capaci y o di e en ia ing in o many o he ypes o cells (see Figu e 2.1). Also, hey possess many di e en cha ac e is ics which enable hem o sel - enew hemsel es inde ini ely and egula e hei popula ion by cell di isions (Kuby, e al. 1992). In humans, haema opoiesis begins in he emb yonic yolk sac du ing 19 he i s wo weeks o de elopmen , by he hi d mon h, hema opoie ic s em cells ha e mig a ed o he oe al li e ; a e ha , he di e en ia ion o HSCs in he bone ma ow becomes he mayo ac o in haema opoiesis. By bi h he e is li le o no haema opoiesis in he li e and spleen (Kuby, e al. 1992, 23). The i s s ep o haema opoiesis consis s in he di e en ia ion o a mul ipo en s em cell, c ea ing ei he a myeloid p ogeni o cell o a lymphoid p ogeni o cell. These cells ha e now los he capaci y o sel - enewal and a e he e o e des ined o gi e ise o cells o i s pa icula cell lineage. Lymphoid p ogeni o s will hen di e en ia e in o Na u al Kille s (NK) cells, T lymphocy es and B lymphocy es. Analogously, myeloid p ogeni o s will gi e ise o e y h ocy es ( ed blood cells, RBCs), pla ele s, basophils, neu ophils and o he immune cells commonly e e ed o as whi e blood cells. Each o hese cells a e c ea ed and induced unde di e en ci cums ances, di e en signals and mic oen i onmen s will p omo e g ow h and di e en ia ion o hese cells. These mic oen i onmen s a e occasioned by sca old like s uc u es c ea ed by s omal cells and by g ow h ac o s ha a i e o hei a ge cells by di usion. To summa ise, i can be said ha he di e en cells ha descend om he HSCs will come o be due o he di e en in e ac ions o he di e en ac o s ela ed wi h i s g ow h and di e en ia ion. Haema opoiesis is egula ed a gene ic le el, he de elopmen o he s em cells in o he di e en cell ypes equi es he exp ession o di e en se s o lineage- de e mining and lineage-speci ic genes a app op ia e imes and in co ec o de (Kuby, e al. 1992, 24). The e o e, we can say wi h ce ain y ha he p o eins exp essed by hese genes a e o u mos impo ance and will de e mine he ou come o cellula di e en ia ion. Much emains o be lea n in his ield; howe e , he e a e some disco e ies ha ha e p o ided a e y use ul insigh in o unde s anding how his p ocess wo ks. One o hese ad ances in he immune-gene ic esea ch led o he disco e y o Leukocy e Adhesion De iciency (LAD), a he i able gene ic disease ha we will discuss in he ollowing pages due o he close ela ionship wi h ou esea ch. 20 2.1.2 Immune Sys em O gans Like many o he sys ems, he Immune sys em has dis inc o gans and issues wi h specialized unc ions, des ined o con ibu e o he de elopmen o immune esponses. Immunologis s classi y hese o gans by unc ion, di iding hem in o p ima y and seconda y lymphoid o gans. The hymus and bone ma ow belong o he cen al o p ima y lymphoid o gans, whe eas he seconda y lymphoid o gans a e comp ised o lymph nodes, he spleen and o he issues. Ma u a ion o lymphocy es akes place in he p ima y lymphoid o gans, whe eas in he p e iously men ioned seconda y lymphoid o gans, he ma u ed lymphocy es in e ac wi h an igens. These lymphocy es will hen ci cula e a ound he body h ough he ci cula o y and lympha ic sys em (Figu e 2.2), de ec ing and igh ing o possible pa hogens. P ima y lymphoid O gans Lymphocy es gene a ed in haema opoiesis a e ye o be ma u ed and become commi ed o an igenic speci ici y. Only a e he cell has ma u ed wi hin a p ima y o gan, is he cell immunocompe en . T-lymphocy es a e called as such due o he ac ha hey ma u e in he hymus, a p ima y lymphoid o gan. The hymus is a specialized o gan o med by specialized cells called hymocy es sepa a ed by connec i e issue. This o gan is he si e o - lymphocy e de elopmen and ma u a ion. The hymus is la and bi-lobed, being loca ed abo e he hea (see Figu e 2.2), i is e y complex and o u mos impo ance. I s ole in he immune sys em can be s udied in mice by examining he e ec s o neona al hymec omy. Thymec omized mice show a d ama ic dec ease in ci cula ing lymphocy es o he T-cell lineage and an absence o cell- media ed immuni y (Kuby, e al. 1992, 41). 21 Figu e 2.2: Schema ic o he Lympha ic Sys em (Blaus 2013). The o he p ima y lymphoid o gan is he bone ma ow. The bone ma ow is a sponge-like issue si ua ed inside o bones; mos o he cells o ou immune sys em a e p oduced and de eloped he e and, once hey de elop and ma u e, hey mig a e o he bloods eam o ci cula e he body. A bi h, many bones con ain ed bone ma ow, which ac i ely builds de ence cells. Du ing he cou se o li e, mo e and mo e o his ed bone ma ow u ns in o a issue. E en ually, all he ed bone ma ow ans o ms, excep in a ew bones, such as he ibs, he b eas bone and he pel ic bone (Schmid , Lang and Heckmann 2010). Lymphocy es ha o igina e om his o gan a e e e ed o as B-lymphocy es. Lympha ic Sys em The ci cula o y sys em is a p essu ised sys em, because o his as blood ci cula es he body, he plasma seeps h ough he capilla y’s endo helium, his luid is called in e s i ial luid and ba hes all he su ounding cells. Due o he high olumes o his luid being pe mea ed h ough he capilla y walls, a lympha ic sys em needs o collec i o p e en swelling o he nea by a eas (edema) which would slowly become li e h ea ening. This lympha ic sys em o ms a ne wo k o lympha ic essels which collec all he lymph (plasma) and hen d ains i back in o he blood. When a o eign an igen gains en ance o he 22 issues, i is picked up by he lympha ic sys em and is ca ied o he lymph nodes whe e i is apped and ea ed acco dingly. 2.2 Leukocy e Ac i a ion and Mig a ion When we expe ience an in ec ion om an ex e nal agen , ou bodies eac in many di e en ways o signal ou immune sys em ha he in eg i y o a issue has been comp omised. An in lamma o y esponse is gene a ed as a esponse o a local inju y o auma, which he immune sys em de ec s as a dis ess signal. This in lamma ion is a complex esponse ha in ol es se e al immune sys em cells and nume ous media o s. Once he inna e immune sys em eaches he a ea o in ec ion, i moun s he ini ial a ack agains he o eign pa hogen. Howe e his ini ial de ence may no su ice and lymphocy es a e ec ui ed and become ac i a ed in esponse o an igens wi h he goal o helping in he ba le agains o eign pa hogens. This p ocess is e y complex he e o e we shall s udy i in mo e de ail in he ollowing pages. 2.2.1 Cell Adhesion Molecules The human body is made ou o many cells and issues join oge he o ming bigge and mo e unc ional s uc u es. All hese cells a e bonded oge he by wha we call cell adhesion molecules (CAMs), and i is hese same molecules ha help he leukocy es and he immune sys em cells in e ac wi h o he issues. Leukocy es a e ci cula ing a ound he body wai ing o an in ec ion, eady o a ack o eign pa hogens. The e o e, ascula endo helium se es as an impo an en y poin o hese leukocy es o a i e a he a ge ed issue. Due o he high p essu e in he ci cula o y sys em, cells a e unde high mechanical s esses and ind i impossible o a ach hemsel es o he ascula endo helium and espass he ascula ba ie . The e o e, hey need some so o aid o mig a e om he blood o he lymphoid o gans o su ounding issues. This p ocess is called ex a asa ion and i happens pa ly due o he p esence o leukocy e speci ic CAMs ha will bind whi e blood cells o he ascula walls. 23 Endo helial cells exp ess leukocy e adhesion molecules, some a e cell speci ic and o he s a e esponse speci ic, e.g. cy okine p oduced du ing an in lamma o y p ocess. All in all, we can say ha ega dless he o igin o he esponse o speci ici y; hese CAMs a e pa amoun in he mig a ion and mo emen o whi e blood cells ac oss he body. In addi ion o hei ole in leukocy e adhesion o ascula endo helial cells, CAMs on leukocy es se e o inc ease he s eng h o he unc ional in e ac ions be ween cells o he immune sys em. Va ious adhesion molecules ha e been shown o con ibu e o he in e ac ions be ween TH cells and APCs, TH and B cells, and CTLs and a ge cells (S ein and Nombela-A ie a 2005). Mos o hese CAMs belong o ou amilies o adhesion p o eins: selec in 1 , mucin-like, in eg in and he immunoglobulin amily. Selec ins The selec in amily o cell adhesion p o ein a e a g oup o glycop o eins ha has a speci ic domain ha enables biding wi h o he molecules due o he a ini y o his lec in-like domain o speci ic ca bohyd a es. The selec in p o ein amily g oups h ee molecules: L-, E- and P-selec in. E- and P-selec in a e mos ly ound on ascula endo helium whe eas L-selec in is exp essed on he su ace o mos leukocy es. E-selec in is a e y in e es ing p o ein ha needs syn hesis o new p o eins o i o exp ess i sel on he ascula walls. These new p o eins commonly appea a e he s imula ion o in lamma o y cy okines. Selec ins a e he ini ial bonde s o leukocy es o ascula endo helium; he e o e hey play a p ominen ole in leukocy e ex a asa ion. Chemokines a e ano he amily o p o eins ele an in leukocy e ex a asa ion, hese molecules a e esponsible o ce ain CAMs ac i a ion, e.g. E-selec in. Consequen ly hey a e a mayo egula o in lymphocy e and leukocy e a ic (S ein and Nombela-A ie a 2005). Chemokines a e ypically induced due o an in lamma ion p ocess, commonly as a esponse o issue in ec ion. These molecules a e o high impo ance because hey essen ially ac i a e E-selec in 1 Due o he impo ance o selec in amily adhesion p o ein in ou esea ch we shall only ocus on his p o ein o he ime being. 24 molecules. In he e en o chemokines no being exp essed, leukocy es would no be co ec ly ec ui ed due o E-selec in no being ac i a ed (Collins, e al. 1991). 2.2.2 Leukocy e Ex a asa ion Lymphocy e and Leukocy e ex a asa ion as a whole is a mul is ep p ocess ha occu s in blood essels and egula es he a ic o whi e blood cells in he body. This p ocess is no mally ini ia ed by an in lamma o y esponse due o he in ec ion o a issue by a o eign agen . Once his in lamma o y esponse de elops, cy okines and o he in lamma o y media o s ac on he local blood essel walls, inducing he exp ession on endo helial CAMs. Once he si e is ac i a ed, leukocy es can hen p oceed o ex a asa e o he si e o in ec ion. Fo his o happen, leukocy es will ha e o s ongly a ach hemsel es o he essel wall o a oid being swep away by he highly p essu ised low o blood. Leukocy e ex a asa ion can be di ided in o ou s eps: leukocy e olling, ac i a ion, a es and mig a ion. We shall ocus on he olling phase o he cell’s mo emen . Rolling is media ed by selec ins; his mo emen is comp ised o a loosely a ached bond ha will bind he cell o he endo helium by a low-a ini y selec in-su ace ca bohyd a e in e ac ion. As we’ e men ioned p e iously, E- selec in is ac i a ed by chemokines, he e o e once hey a e p ope ly exp essed in he endo helium hey can adhe e o ca bohyd a es exp essed in leukocy e memb anes. Being such a weak adhesion, leukocy es will only bond b ie ly and soon de ach i sel due o he shea o ce o he ci cula ing blood. Once his happens i will apidly a ach i sel o ano he endo helial cell. This umbling o he cell is epea ed o e and o e , c ea ing a olling mo ion (see Figu e 2.3). This p ocess is ai ly slow; he e o e i slows down he speed o he cell jus enough o allow in e ac ions be ween chemokines p esen in essel wall and he ecep o s on he leukocy e su ace o in e ac and gene a e a igh e bond ha will comple ely adhe e he cell o he wall. Once he mo emen o he leukocy e has been s opped by his igh adhesion, he cell will squeeze h ough wo neighbou ing endo helial cells and a e se he endo helial ba ie and en e he in ec ed issue (Ande son and Ande son 1976). 25 Figu e 2.3: S eps in Leukocy e (Neu ophil) ex a asa ion (H. Shaz, R. S owell y D. Hillye 2011). 2.2.3 Leukocy e Adhesion De iciency Leukocy e Adhesion De iciency o LAD is a gene ic diso de ha is cha ac e ized by a poo leukocy e adhesion o he capilla y walls, making i impossible o he immune sys em cells o adhe e p ope ly and he e o e no ex a asa e o he in ec ed issue. This disease esul s in he immunode iciency o he pa ien and concludes in he pa ien ha ing ecu en in ec ions. LAD a ec ed pa ien s will su e om bac e ial in ec ions om a e y ea ly age and can become a li e h ea ening condi ion due o he in an ’s inabili y o comba o eign pa hogens. This inabili y o comba bac e ia and o he agen s is due o he non-exis ing leukocy e mig a ion. Besides he no able absence o a de ence sys em, LAD pa ien s will also ha e high coun s o leukocy es in he blood and will no be able o o m pus (Abbas, Lich mann and Pillai 2012). As we speak he e is no cu e o his disease, he only cu en cu a i e he apy is in ensi e an ibio he apy o haemopoie ic s em cell ansplan . I wan ed o include his sec ion in his chap e because al hough he e is no cu e, he p o o ype and expe imen ha we p opose in his wo k could be used as a ool o conduc expe imen s and assays o design a no el d ug and, e en ually, es i s sui abili y o cu ing his disease. 32 3.2, we can clea ly see his beha iou in a s aigh channel. In his igu e, he low in he cen al sec ion has a maximum eloci y as opposed o he la e al sec ions whe e a subs an ial dec ease in eloci y is obse ed. Figu e 3.2: Pa abolic p o ile in a luid low simula ion (COMSOL). This beha iou dis up s he way in which we calcula e he speed o he low. Howe e h ough ma hema ical analysis we can ex apola e he equa ion ha go e ns he low. 𝑢(𝑟)=2 𝑉𝑎𝑣𝑔 (1 −𝑟2 𝑅2) , (3.6) 𝑉𝑎𝑣𝑔= −𝑅2 8𝜇 (𝑑𝑃 𝑑𝑥) , (3.7) whe e 𝑟 equals he dis ance om he middle poin and 𝑅 is he adius o he pipe he luid lows h ough. 3.6 Hyd odynamic Focusing Hyd odynamic ocusing is also called low ocusing o shea h low, howe e many names i may ha e, hey all ha e one unde lining unc ion: o o ce a luid o cell o pass h ough a speci ied sec ion. This echnique has been used o many yea s in he ealm o luid dynamics o con ol he way we in e ac wi h he di e en phases o a luid in a closed en i onmen . Hyd odynamic ocusing has 33 been widely used in many applica ions as in low cy ome y (Shule , A is and Tsuchiya 1972). Figu e 3.3: Flow ocusing s uc u e. A low ocusing de ice is mainly composed o h ee inle s and one ou le as seen in Figu e 3.3. The low ocusing me hod has he goal o placing a s eam o luid o solid phase ma e ials in he cen al po ion o he channel by he use o he auxilia y side channels. When he cen al channel which lows a a lowe p essu e, a i es a he junc ion poin , he luid ha comes in om he auxilia y inle s pushes he cen al in e ace in o a de e mined sec ion, which will be ixed by he p essu e a which he ocusing luid is lowing a . This phenomenon and ool widely used in mic o luidics is due o he conse a ion o Ene gy and Mass law. 3.6.1 Conse a ion o Ene gy Ene gy wi hin a luidic sys em emains cons an in such a way ha ene gy ac ing on he sys em is con inuously changed in o o he o ms such as wo k. Fo luids he equa ion ha go e ns his p inciple is e y complica ed, due o ac ha is aken in conside a ion se e al o ms o ene gy ac ing and dissipa ing ou o a de ined body. In i s simples o m i can be desc ibed as: ∆𝐸=𝑐𝑜𝑛𝑠𝑡𝑎𝑛𝑡 . (3.8) This has ce ain ami ica ion such as he conse a ion o olume ic low a e. 𝑄(𝑣𝑜𝑙𝑢𝑚𝑒𝑡𝑟𝑖𝑐 𝑓𝑙𝑜𝑤 𝑟𝑎𝑡𝑒)=𝑣· 𝐴 , (3.9) whe e 𝑣 is he eloci y o he low in 𝑚/𝑠 and 𝐴 is he c oss sec ional a ea. 34 This o mula explains ha in he p esence o a bo leneck, he c oss sec ional a ea will dec ease and he e o e inc ease he speed a which he low is lowing. 3.7 Lab on a Chip Lab on a Chip o “LOC” is a e m used o desc ibe a speci ic use o mic o luidics. As explained in he in oduc o y sec ion o his chap e , mic o luidics came o be due o he need o ha ing a quick and small ield deployable de ice o de ec ce ain chemical agen s in he ai . This ini ial goal o minia u iza ion has been ex apola ed o o he applica ions c ea ing he ield o LOC. LOC ies o syn hesize all he necessa y analy ical es s ha would be needed o pe o m ce ain labo a o y analysis on a silicone chip he size o a coin as seen in Figu e 3.4. Lab on a chip mic o luidic de ices a e being de eloped o be used in many di e en indus ies, wi h he goal o making chemical analysis much as e , cheape and p ecise (Whi esides 2006). One o he said indus ies is heal hca e. Hospi als pe o m housands o es s on a daily basis, cos ing hem a lo o esou ces and money on pe sonnel and equipmen . LOC de ices o e a e y p omising u u e whe e ce ain clinical es ing can be made accessible and a o dable o hose heal hca e acili ies ha canno a o d adi ional glasswa e labo a o ies. Figu e 3.4: Example o mic o luidic lab on a chip de ice. This de ice aims o pe o m chemical analysis using mic o luidic and MEM echnology (Shu e s ock). 35 3.7.1 O gan on a chip Ano he eme ging a ea whe e mic o luidics is being used would be biological emula ion. Labo a o ies wo ldwide a e ying o eplica e he phenomena ha occu in ou bodies wi h he use o mic o luidic echnology (see Figu e 3.5). These de ices a e e e ed o as “O gans on a chip”. Wi h hese o gans on a chip, esea che s aim o s udy how ou body wo ks and how hey could beha e unde he use o ce ain d ugs and diseases. Figu e 3.5: Emula e Lung on a chip mic o luidic de ice. This de ice aims o p o ide a medium in which we es new he apy d ugs. 37 Chap e 4: Simula ions 4.1 In oduc ion Compu e simula ions and CAD so wa e has become an inc easingly used echnology in enginee ing, due o he ad an ages o wo king in a i ual en i onmen . This chap e con ains he simula ions pe o med on COMSOL ha we e necessa y o he de elopmen o his esea ch. Simula ions we e used and a e needed in his esea ch because hey pose a cheap and accessible en i onmen whe e we can pe o m es s on ou p o o ypes be o e building he physical models; hese models gi e a deep unde s anding o he possible beha iou o ou de ice. The so wa e o choice was COMSOL, a Mul iphysics modelling so wa e which allows simula ing mic o luidic and c eeping lows. 4.2 Simula ions 4.2.1 Fluid Flow Simula ion This sec ion co esponds o he simula ion o a iscous luid along a mic o luidic channel in a lamina low. The c oss sec ion o he channel is 50 μm x 100 μm. 38 Figu e 4.1: Fluid Flow simula ion 50 μL/h XZ plane isualiza ion. The uni s exp essed in he legend a e m/s. As we can obse e in Figu e 4.1, whe e eloci y con ou s a di e en longi udinal loca ions a e show, he low is as e in he cen al po ion a he han on he sides, his is he well-known e ec o he symme ical pa abolic luid p o ile. The a e age eloci y is gi en as: 𝑣𝑚=1 4𝜂 ∆𝑃 ∆𝑥 𝑅2 , (4.1) whe e, 𝜂 is he dynamic iscosi y, ∆𝑃 he di e ence o luid p essu e, ∆𝑥 he dis ance co e ed and 𝑅 hal he wid h o he channel. While he eloci y a a gi en poin is de ined by he ollowing o mula: 𝑣(𝑥)=𝑣𝑚 [1 −𝑥2 𝑅2] , (4.2) whe e 𝑣𝑚 is calcula ed in Fo mula 4.1, 𝑅 is hal o he wid h o he channel and 𝑥 is he posi ion along he pe pendicula axis o he low o he luid. This e ec o as e luid a he cen e o he geome y is gi en by he wall ic ion wi h he lowing luid. 39 Figu e 4.2: Fluid Flow simula ion 50 μL/h YX plane isualiza ion. The uni s exp essed in he legend a e m/s. As we can obse e in Figu e 4.2 and Figu e 4.1 whe e he low is as e in he cen al sec ion, his e ec is gi en in bo h he 𝑥 and he 𝑧 di ec ion, his is due o he p esence o side walls in bo h hese axes. In he ollowing igu e (see Figu e 4.3) we can pe ec ly obse e how he speed a ies in he di e en 𝑥, 𝑦 and 𝑧 coo dina es. Due o he lamina low and dynamic iscosi y he p o ile o ou low will be pa abolic. Fo his simula ion we used pa icle acing physics o clea ly ep esen he di e en speed and hei dis ibu ion along he channel. 40 Figu e 4.3: Pa abolic p o ile o a lamina low in a ec angula channel. 4.2.2 Shea h Flow Simula ion These ollowing simula ions y o show he e ec o he shea h low in a mic o luidic channel. In Figu e 4, he o al low in he cen al, pos ocusing s age is 50 μL/h; in his plo all h ee inle s ha e he same low a e: 16.67 μL/h, as gi en by he ollowing o mula. 𝑄𝑡𝑜𝑡𝑎𝑙 =𝑄1+𝑄2+𝑄3 . (4.3) 𝑄1, 𝑄2 and 𝑄3 co espond o he di e en inle s o he mic o luidic hyd odynamic ocusing de ice; by al e ing he di e en in low a es o hese inle s we will ha e di e en ocusing wid hs in he pos - ocusing sec ion. 41 Figu e 4.4: Su ace plo o he luid eloci y. Speed measu ed in m/s. In Figu e 4, we can obse e he su ace plo o he luid eloci y. Each o he inle s ha e he same low a e: 4.63 x 10-12 m3/s o 16.67 μL/h. The o al low a e in he cen al channel is 50 μL/h. We immedia ely see ha in his cen al channel, he eloci y magni ude is much g ea e . Flow a e in a pipe is gi en by luid speed mul iplied by he a ea o he channel c oss-sec ion: 𝑄=𝑣𝑎𝑣𝑒𝑟𝑎𝑔𝑒 ×𝐴𝑐𝑟𝑜𝑠𝑠−𝑠𝑒𝑐𝑡𝑖𝑜𝑛 . (4.4) 𝑣𝑎𝑣𝑒𝑟𝑎𝑔𝑒 =𝑄 𝐴𝑐𝑟𝑜𝑠𝑠−𝑠𝑒𝑐𝑡𝑖𝑜𝑛 . (4.5) The e o e, while he c oss sec ional a ea emains cons an we see a h ee old inc ease in olume ic luid low a e. 48 5.3 Mic o ab ica ion Technology Fo he de elopmen o his mic o luidic de ice, he s anda d mic o luidic p o ocol was used. Fi s ly, a pho omask was de eloped using mic o ab ica ion echnology, hen se e al Silicon wa e s we e p oduced by pho oli hog aphy, las ly, PDMS was pou ed on on o he Silicon wa e s and cu ed o p oduce he esul ing mic o luidic channel. 5.3.1 Elec on Beam Li hog aphy As i is he case wi h almos all o mic o luidic echnology, his echnology was i s ly de eloped o elec onics applica ions, howe e due o he capaci y o achie ing e y high esolu ions i may be used in se e al di e en applica ions. This echnique consis s in pe o ming pa e ns on a subs a e using an elec on beam. This pho omask is hen used in he successi e s ages o he de elopmen o mic o luidic channels. The pho omask was de eloped a he Uni e si y o Tokyo, Takeda Tipezaki Building Clean Room by he s uden Sayaka Nomu a o he Tokyo Uni e si y o Science. 5.3.1.1 Pho omask C ea ion Figu e 5.2: Elec on Beam Schema ic showing he p ocess o cha ac e iza ion o a pho omask. As illus a ed in Figu e 5.2, he di e en s eps o making he pho omask a e: 1. Ini ially, he pho omask (see Figu e 5.2) is co e ed in a special pho o esis ha when in con ac wi h he elec on beam will d aw he desi ed pa e n on o he mask (see Table 5.1). This pa is c i ical in he de elopmen o he mask, 49 an inco ec exposu e o he pho o esis will conclude in an inaccu a e mas e pho omask ha will hen in luence in a nega i e manne he es o he p ocess. Fo his sec ion o ou p ocess he machine shown in Figu e 5.3 was used, wi h he pa ame e s shown in Table 5.2, espec i ely. Each esis has speci ic cu ing pa ame e s and a equi ed ene gy pe uni a ea, he e o e he amoun o ligh and exposu e ime mus be adjus ed acco dingly o he esis used. An inco ec adjus men o hese speci ica ions could esul in an insu icien ch omium emo al o he unde lying laye s. Manu ac u e Toppan Model ST-TLR6-TQZ-5009(5T) Mask dep h 2.3 mm Resis ZEP7000 Resis dep h 3000 Å P ebaking 190 ºC o 30 minu es Me al laye C / C O2 Me al Laye dep h 100 nm Table 5.1: Pho omask Speci ica ion. Figu e 5.3: Elec on Beam Li hog aphy equipmen used o he c ea ion o he pho omask. This sys em is loca ed in Tokyo Uni e si y. Manu ac u e Ad an es Model F5112 Smalles dimension possible 100 nm Dimension de ia ion 3σ ≤ 15 nm Supe posi ion p ecision |mean alue| + 3 σ ≤ 40 nm Table 5.2: Speci ica ion o he Ad an es Elec on Beam Sys em. 50 2. Once he esis has been exposed o he elec on beam, i has o be de eloped and hen insed o . Isop opyl alcohol (IPA) and me hyl isobu yl ke one (MIBK) a e he chemicals used in his p ocess. The machine EVG 101D Au oma ic De elope was used o his s ep (see Figu e 5.4) (see Table 5.3). Figu e 5.4: Au oma ic pho omask de eloping sys em. Loca ed in Tokyo Uni e si y. Manu ac u e EVG Model 101D Spin module max. deploymen num. 1 Types o De elopmen P essu e ank, low con ol, ni ogen nozzle o spay de eloping, paddle de eloping, inse de eloping Table 5.3: Au oma ic Pho omask De elope echnical Speci ica ions. 3. This s ep is called E ching. E ching consis s in using s ong acids and chemicals o cu o p oduce a pa e n in o he unp o ec ed pa s o a me al su ace. In ou case, we imme se he mask in o a chemical solu ion wi h he goal o emo ing he exposed me al and hus o ming ou pa e n. In some cases Plasma Ashing is used as a me hod o p e- ea he su ace o a be e e ching p ocess. Using a plasma sou ce, Plasma Ashing is pe o med on o he pho o esis o enable a be e pene a ion o he chemicals used in he la e e ching p ocess. The ashing appa a us used was he FA-1 manu ac u ed by Samco (see Figu e 5.5) (see Table 5.4). The e ching appa a us used was he Fai child 1140 e ching machine (see Figu e 5.7) (see Table 5.5) 51 Figu e 5.5: Ashing appa a us loca ed in Tokyo Uni e si y. Manu ac u e Samco Model FA-1 Gas CF4 (50 sccm) O2 (50 sccm) Maximum RF 200 W Table 5.4: Ashing appa a us echnical speci ica ions. 4. Finally, we wash o he esis wi h an ashing de ice (see Figu e 5.6); consequen ly, all he esis is emo ed om ou mask lea ing behind a Ch omium Oxide laye on op o a glass subs a e, wi h a pa e n ha will hen be used h oughou he whole mic o luidic de ice ab ica ion. This p ocess p e iously explained will lead o he c ea ion o a pho omask (see Figu e 5.6). The pho omask has a se ies o pa e ns e ched in o he glass-me al su ace which a e anspa en , his will enable ligh o pass h ough and hus c ea e he shape o he de ice we desi e. Figu e 5.6: Pho omask used in he mic o luidic channel de elopmen , c ea ed by Sayaka Nomu a in 2015. 52 Figu e 5.7: E ching appa a us, loca ed in Tokyo Uni e si y. Manu ac u e Fai child Model 1140 Ou pu Powe 3.6 kW Supply P essu e CDA 80PSI N2 60 PSI D1wa e 30PSI Discha ge Condi ion 80SCFM 20SCFM Table 5.5: Technical speci ica ions o he e ching machine. 5.3.2 Pho oli hog aphy We call pho oli hog aphy he p ocess in which we use UV ligh o cu e pho osensi i e chemicals in o de o make pa e s on a wa e . Jus like he p e ious echnology, his p oceeding was bo owed om he elec onics ield. In elec ic and elec onic enginee ing, pho oli hog aphy is used in he manu ac u ing o ansis o s and complex in eg a ed ci cui s. In his p ocess, we shall use he pho omask ab ica ed in he p e ious sec ion and use i o pa e ning o a silicon wa e , his silicon wa e will be he mas e mould we will hen pou ou PDMS solu ion on o build he channel. 53 5.3.2.1 Silicon pa e ning p ocedu e Figu e 5.8: Mas e mould li hog aphy p ocess. As illus a ed in Figu e 5.8, he di e en s eps o manu ac u ing he silicon wa e mic o luidic channel mas e mould: 1. The i s s ep in pho oli hog aphy is o apply a coa ing o pho o esis o ou silicon wa e . In ou case we used SU-8 pho o esis (see Figu e 5.9) due o i s chemical a ibu es (see Table 5.6). Figu e 5.9: SU-8 pho o esis . Manu ac u e Mic o Chem. Model SU-8 3000 Adhesi e s eng h 71 MPa Glass ansi ion poin 198 ºC, DMA an δ Volume Resis i i y 1.8 x 1016 Ω·cm Wa e Abso p ion 0.5 % 85 ºC / 85 %RH, 120h Table 5.6: Chemical Speci ica ions o Su-8 pho o esis . 54 In o de o apply he pho o esis we i s pou he solu ion on o a silicon wa e and we spin i a a a e o 2250 pm (see Table 5.7) wi h he use o he spin coa e (see Figu e 5.10), his spinning will ex end he SU-8 o e he en i e silicon wa e achie ing a hickness o 50 μm. We hen p ebake he silicon wa e using a ho pla e a 60 ºC o 1 minu e and hen a 90 ºC o 15 minu es. A e hea ing, we lea e o cool down in oom empe a u e. Figu e 5.10: Spin coa ing appa a us loca ed in Mo lab, Tokyo Uni e si y o Science. Manu ac u e Ac i e Model ACT-220D Ro a ion ange 200 ~ 8000 pm Ro a ion e olu ions ± 3 pm Vacuum 350 To S eps / pa e ns 10 s eps / 100 pa e ns Table 5.7: Technical speci ica ions o spin coa e . 2. The nex s ep consis s o exposing he spin coa ed silicon wa e o a UV ligh o gene a e he pa e n on ou wa e . Fo his we use he pho omask aligne (see Figu e 5.11). This machine will expose ou silicon wa e o a UV sou ce wi h he shape o he pho omask, o his o happen UV ligh is emi ed h ough he sou ce (see Table 5.8) and only passes h ough he pho omask in he a eas whe e he Ch omium Oxide has been e ched ou in he p e ious p ocess. Once he pho o esis is cu ed wi h UV ligh , we can p oceed o he de elopmen o he SU-8. A e he exposu e, we pe o m a pos -bake by hea ing on a ho pla e he Si wa e a 60 ºC o 1 minu e and 90 ºC o 15 minu es. 55 Figu e 5.11: Mask Aligne appa a us, loca ed in Tokyo Uni e si y o Science. Manu ac u e Nano ec Model ES20 Exposu e a ea 105 mm Illumina ion uni o mi y ±5 % Illumina ion dis ance 200 mm Dominan Wa eleng h 365, 405, 436 nm UV i adia ion s eng h Mo e han 35m W/cm2 (a 365 nm) Table 5.8: Technical speci ica ions o Nano ec mask aligne . 3. Finally, we p oceed o de elop he silicon wa e by insing and imme sing he wa e in o SU-8 de elope . In some cases, SU-8 will emain; he e o e, we will wash he wa e wi h isop opanol, ace one, e hanol and dis illed wa e . Once we ha e comple ed all his p ocess we will ha e co ec ly de eloped a silicon wa e wi h ou pa e n d awn on i (see Figu e 5.12). Figu e 5.12: Silicon wa e mic o luidic channel mas e mould. 56 5.3.3 PDMS Channel Fo he cha ac e isa ion o he mic o luidic channel, we used PDMS (Figu e 5.13). PDMS is a widely used silicon-based o ganic polyme in mic o luidics; his polyme is used due o i s many ad an ages. As a esul o i s lexibili y and i s mechanical p ope ies his ype o echnological p ocedu e is also e e ed o as so li hog aphy. Figu e 5.13: PDMS. In o de o c ea e ou PDMS channel, we will simply pou he mix u e o PDMS on ou SU-8 mould and lea e o d y up un il o ming a solid block. The mix u e we pou is a 10:1 PDMS o cu ing agen a io (Table 5.9). No e ha be o e pou ing he PDMS on o he wa e we should place he mix u e in o a acuum o 40 minu es o emo e all bubbles. Manu ac u e Dow Co ning To ay Model Silpo 184 Viscosi y 5000 cS Densi y 1.11 g/cm3 Mixing Ra io Main Agen : Cu ing Agen 10:1 Hea ing empe a u e and ime 60 ºC 80 minu es, 150 ºC 30 minu es Re ac i e Index 1.41 Table 5.9: PDMS chemical speci ica ions. Once we pou he PDMS we hea a 60ºC o 80 minu es and 150 ºC o 30 minu es espec i ely. When d y (see Figu e 5.14), we can simply cu wi h a azo blade, punch holes in o he inle and ou le s and place on a clean glass slide o pe o m ou expe imen s and es s. 57 Figu e 5.14: Final mic o luidic PDMS channel be o e assembly. 5.4 E-Selec in subs a e p epa a ion The cell adhesion molecule used in his wo k is he E-selec in (see Figu e 5.15) he e o e we mus coa he subs a e o he channel o ou se up o wo k p ope ly. The p epa a ion me hod is ai ly simple due o he cha ac e is ics o he ma e ials used (see Table 5.10). Figu e 5.15: E-selec in. Fi s we coa ou glass slide wi h a ine laye o PDMS, o his we mus spin coa he PDMS on he glass subs a e. Thanks o he unc ional amino g oup o he E-selec in, we can easily bond he CAM o he PDMS o ming a pep ide bond, which s ongly binds he molecule o he subs a e o ou mic o luidic channel. The e o e, we mus simply pou he E-selec in (500 mg) dissol ed in 64 6.1.3 Discussion As we’ e seen p e iously, he se up and execu ion o his expe imen show a p omising hyd odynamic ocusing. We ha e demons a ed ha by a ying he low a es in each o he inpu s we can modi y he wid h o ou ocused channel. This is a undamen al i s s ep in he de elopmen o ou de ice, conside ing ha we can now con ol he amoun o luid and cells ha will pass h ough he cen al po ion o ou channel. We mus ake a minu e o poin ou he possible e o s in calcula ing he size o he shea h. The e o in he calcula ions may be o ±1 μm; his is no signi ican as he size o he leukocy e is gene ally 10 μm. When using he inal i e a ion o ou design, he use may wan o change he condi ions o low in he channel he e o e we can make many combina ions o low a es o i ou needs. In ou case he since we wan a single cell ocused low, we aim o ha e dimensions anging om 11 μm o 15 μm he e o e we could use he abo e es ed combina ions. When choosing said low a es, we mus elec app op ia e a es bea ing in mind he o al low downs eam om ocusing poin . Once he h ee lows mee in he channel he o al low a e would be he sum o he h ee low a es, as indica ed in he ollowing equa ion: 𝑄=𝑄𝑐𝑒𝑛𝑡𝑟𝑎𝑙 𝑐ℎ𝑎𝑛𝑛𝑒𝑙 +2×𝑄𝑠𝑖𝑑𝑒 𝑐ℎ𝑎𝑛𝑛𝑒𝑙𝑠 . (5.3) As we know, cells will beha e di e en ly wi h di e en lows; he e o e, we mus adjus hese pa ame e s acco dingly. 6.2 Rolling Cell Expe imen In his second expe imen , we ec ea e he olling cell expe imen on a simple s aigh channel mic o luidic de ice. The goal o his expe imen is o es he olling condi ions in which he las expe imen will ake place. We shall expe imen unde he same condi ions as in he las expe imen and expose he p oblems we ace when wo king wi h he absence o a hyd odynamic ocusing module. 65 6.2.1 Expe imen al Se up Figu e 6.4: Schema ic image showing he expe imen al se up o his assay. The se up is e y simila o he p e ious expe imen , changing in aspec s such as he numbe o pumps used and he na u e o he luid used. Due o he ac ha we a e es ing li e leukocy es on his assay, we used PBS as wo king luid in o de o keep he medium as s able as possible. As explained in chap e 6.4, his expe imen needs an E-selec in coa ed su ace o he leukocy es o bind co ec ly; he e o e a p e iously ea ed subs a e was used o his expe imen . The condi ions, as explained p e iously, do no change much om he p e ious expe imen ; howe e we used Calcein AM o his expe imen o s ain he cy oplasm o he cell. Calcein AM is a luo escen dye wi h emission wa eleng hs o 495/515 nm. This dye is used in biology due o i s abili y o pene a e he cellula memb ane in li e cells, making i a e y use ul ool o es ing cell iabili y and o sho e m cell labelling. 6.2.2 Resul s The ollowing esul s we e ob ained a 0.2 Pa o wall shea s ess and a e depic ed in Figu e 6.5 and Figu e 6.6. As we can obse e only leukocy es on he side o he channels a ach, whe e he luid low is slowe , lea ing he cen al sec ion comple ely ba en. 66 Figu e 6.5: Top iew o mic o luidic channel. Leukocy es low on he edges o he channel as opposed o he cen al a ea whe e we would expec hem. Figu e 6.6: Top iew o mic o luidic channel. Leukocy es low pa h is sca e ed h oughou he whole domain o he channel. 6.2.3 Discussion As we can see in he Figu es 6.5 and 6.6, he condi ions in which he measu emen s ake place a en’ he ideal posi ion o us o ha e an accu a e eading on he esul s o he expe imen . This is he eason we need a shea h low, in o de o ocus he lymphocy es in he cen al domain o con ol all he a iables in ou u u e es s. Besides his incon enience, he se up wo ks as expec ed: he leukocy es bind o he subs a e and oll a he app op ia e speeds. low 50 μm low 50 μm 67 6.3 Rolling Cell Shea h Flow Expe imen In his expe imen we es he de ini i e e sion o ou expe imen in o de o alida e he p o o ype we ha e c ea ed. We shall in oduce a sy inge o PBS wi h li e lymphocy es suspended and p oduce a shea h low o cen e he immune cells in he channel o ha e mo e con ollable in e ace. 6.3.1 Expe imen al Se up The expe imen al se up is simila o hose explained in he abo e expe imen s. The subs a e was p epa ed he day be o e and le o e nigh in he incuba o a 37.5 ºC, as explained in chap e 5. The channel used was p epa ed on he day o he expe imen ollowing he p ocedu e o so li hog aphy explained in chap e 5. Once he channel was p epa ed i was s e ilized acco dingly wi h e hanol and dis illed wa e ollowing he same p o ocol as he expe imen s explained p e iously. The sy inges used o he expe imen s con ained PBS alone and PBS wi h lymphocy es suspended in hem, espec i ely, p epa ed ollowing he same s eps as he p e ious expe imen s. The lymphocy es dyed wi h Calcein AM, in he same way we pe o med in he p e ious expe imen . 6.3.2 Resul s The esul s obse ed in his expe imen we e as expec ed. When he lymphocy es we e in oduced in o he mic o luidic channel, he cells we e ocused in o he cen al sec ion o he pos e io channel. This is due o he ac ion o he hyd odynamic ocusing module we ha e buil . As we can see in he p e ious expe imen and on Figu e 6.5, we can obse e ha he low o he leukocy es is andom, he same happens in Figu e 6.7(a) and Figu e 6.7(b). Howe e once he cells ha e been ocused by he ac ion o he shea h low hey ci cula e h ough he cen e o he channel as obse ed in he ollowing images o Figu e 6.7(c)-(h). 68 Figu e 6.7: Time-lapse isualizing he low o he lymphocy es h ough he low ocusing module. The o al ime o his ideo las s 1 second. The leukocy e can be obse ed as a whi e do o e a g ey backg ound. This image was ex ac ed using luo escence mic oscopy. We ha e o bea in mind, ha al hough we a e ocusing he low o he lymphocy es in he cen e o he channel, due o he physics o he hyd odynamic ocusing sec ion, he speed a which hese cells mo e along he channel will also inc ease. In his said sec ion, we a e pumping h ee imes he olume o luid as pumped in he ini ial sec ion he e o e as we explained in chap e 3, he inc ease o olume being pumped unde he cons an wid h o he channel will cause he speed o he pa icles o be inc eased. 6.3.3 Discussion The ini ial esul s we e posi i e, and we managed o ob ain he esul s we we e looking o . The ul ima e goal was o c ea e a s eam o ocused cells in o de o analyse hem in a mo e con olled en i onmen . Howe e good he esul may be, he e a e ce ain aspec s we mus alk abou ega ding his expe imen . Fi s ly, as we ha e obse ed in he esul s, he speed o he cells inc eases by a ce ain ac o depending on he low in he inle s. The speed o he pa icle will see i sel inc eased depending on how ocused he low o cells a e. Meaning ha he mo e ocused we desi e ou model o be he highe he speed will be in ou channel, due o he low o highe olumes pe uni o ime pumped in o he (a) (b) (c) (d) (e) ( ) (g) (h) 69 sys em. This may pose ce ain p oblems, due o he limi a ions o he equipmen used o he expe imen . I we desi e a ce ain low pa icle speed (whe e Q ≤ 20 μL/h) whe e we aim o pe o m an expe imen wi h a speci ic p essu e ac o and we wan a high ocusing a e, he cen al inle s mus pump he expe imen al luid a e y low lows, meaning ha he mic o luidic pumps used migh ha e ce ain limi a ions. When pe o ming high ocusing a es and lowe pa icle speed expe imen s, he mic o luidic pumps exhibi ed echnical limi a ions and gene a ed a pulsa ion e ec in he luid. This pulsa ion is a e y nega i e e ec ha we wan o e adica e a all cos s. Secondly, we obse ed ha he binding wi h he E-selec in subs a e was no always achie ed and suspec his is due o he absence o con ac wi h he subs a e o he channel. Due o his mos o he cells will low along he channel ne e a aching o he subs a e. Howe e no hing can be done, because in he e en o o cing he cells o a ach we mus exe a o ce on o he lymphocy e ha would modi y he beha iou o he cell signi ican ly. All in all, one hing is clea , al hough he expe imen has gi en us posi i e esul s, luck is also a ac o when pe o ming expe imen s because lymphocy es can ail o a ach as discussed p e iously. 6.4 Pulsa ion Expe imen In his expe imen we aim o obse e a wha low he mic o luidic pump, Ha a d Appa a us Pump 11 Eli e, exhibi s mechanical limi a ion and hus a ec he low o ou luid causing pulsa ion. 6.4.1 Expe imen al Se up Fo his expe imen we ab ica ed a simple s aigh channel mic o luidic chip and pumped in o he channel mic o pa icles suspended in a dis illed wa e solu ion a lows unde 5 μL/h as seen in Figu e 6.8. We hen obse ed he low o he luid unde he luo escence mic oscope and analysed he esul s. 70 Figu e 6.8: Schema ic image showing expe imen al se up o his sec ion. 6.4.2 Resul s A e es ing a ange o di e en lows, we obse ed pulsa ion in lows unde 5 μL/h. 6.4.3 Discussion As poin ed ou in he esul s sec ion, pulsa ion occu s in lows unde 5 μL/h, indica ing a limi a ion in he appa a us used in ou expe imen a ion. The e o e, when pe o ming expe imen s ha equi e low low a es, we mus be e y ca e ul no o su pass his alue because i could in oduce undesi ed al e a ions o ou expe imen ha could change he esul s o ou es s. 71 Chap e 7: Conclusion 7.1 Conclusions A e analysing all he ele an esul s om he di e en expe imen s we can conclude he ollowing:  The po en iali y o mic o luidics o biomedical esea ch has been p o ed. Mic o luidics poses as an excellen echnology ha is widely used in many labs a ound he wo ld.  The impo ance o mul iphysics simula ion so wa e in mic o luidic esea ch has been con i med.  A mic o luidic sys em o leukocy e expe imen a ion o Lab on a Chip ha o e comes he ini ial non cen al olling p oblem has been success ully de eloped.  Al hough he sys em sol es many p oblems, in cases o low low a es, he sys em p esen s limi a ions due o he equipmen used. 7.2 Fu u e Wo k Al hough he sys em ha has been designed is ully unc ional and could be used in u u e esea ch, he e is s ill oom o imp o emen . The ollowing ad ances a e p oposed as a way o imp o ing he capabili ies o he sys em.  Con inue pe o ming es s o e alua e unc ionali y.  Pe o m olling cell adhesion analysis ia Nomu a´s so wa e o compa e he nume ical da a.  Expe imen wi h di e en leukocy es and cells.  Expe imen wi h ci cula ing cance cells.  De elop a new subs a e wi h li e epi helial human cells o u u e expe imen s. 73 Bibliog aphy Abbas, Abul K., And ew H. Lich mann, and Shi Pillai. Cellula and Molecula Immunology. 7 h ed. Philadelphia: Else ie , 2012. Ande son, A. O., and N. D. Ande son. “Lymphocy e emig a ion om high endo helial enules in a lymph nodes.” Immunology 31, no. 5 (1976): 731. Blaus, B uce. Lympha ic Sys em. N.A., 5 Sep embe 2013. Collins, T., e al. “S uc u e and ch omosomal loca ion o he gene o endo helial-leukocy e adhesion molecule 1.” Jou nal o Biological Chemis y 266, no. 4 (1991): 2466-2473. Day, Michael A. “The no-slip condi ion o luid dynamics.” E kenn nis 33, no. 3 (1990): 285-296. 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