rmt 2006

microfabrication

fabricating small patterns or structures in micrometer. (not about the accuracies or tolerances) (upper is 100 microm and lower 100 nm)

microstructure technology

looking at fabrication, mechanics, fluidics and optics.

• focus on properties and behavior of materials at microscope scale

• understanding and optimizing materials for engineering and manufacturing purposes

• involves techniques to modify or observe material properties at microscale—> enhance prop

microsystem technology (MST)

Micropatterns, structures, sensors, actuators, transducers, energy sources and packaging.

• focuses on miniatured systems that integrate different functions at micom scale or even smaller

• designing and fabricating complex systems that integrate various components (applications such as medical devices)

• microfrabriation and integration of small-scale devices

microfabrication

making already existing structures smaller, but also produce meta-materials. Materials with unfound properties in the nature.

• pulling at one side and the other side also expending

scale influence on physica

volume shrinks faster than areas, areas shrink faster than lines—> influences the corresponding forces

microfabrication where does it take place

happens in clean room

• controlled temp+ humidity

• low partile concentration

• low vibrations

• yellow light

• air filtering

—> takes place in vacuum

—> takes place in plasma

ways of air filtering

• laminar: parallell smooth flow, no mixing, low velocity, and Re also low, viscous is dominant and flor pattern is predictabe ISO<6 and for high precision

• turbulent flow: moves chaotic and irregular flow is highly disordered and irregular fluctations. A high velocity, Re is found and inertia dominates and is unpredicted (higher ISO)

Light is sometimes blocked two zones

low transmission: below 500 nm to ensure high contrast between exposed and non exposed photoresist parts

high transmission: above 550 nm, allows energy-efficient clean room illumination at reasonable visibility

why vacuum need?

due to avoding entrapment of air in molded area/materials. Preventing contamination and enable collusion-free propagation of deposition material towards substrates that need to be coated. Preventing oxidative degredation, and absorption of high radiation.

microfabrication in plasma, what is plasma?

Plasma is a state of matter whereby it is made out of ions and free electrons. They are electrically conductie and made by heating neutral gas or subjecting it to strong electromagnetic fields (plasma etching or depositioning)

plasma etching

process whereby material is removed fro surface typically thin film or substrate by exposing to plasma (used for patterning or etching)

plasma based depositioning

used to deposit thin films or materials onto a substrate, the plasma facillitates a chemreaction between the fases on the surface

different types of microfabrication

etching, photolithography, sputtering, chemical vapor depositioning, atomical depositioning, nanoimprint lithography and electroplating

standard photo-optical/ UV lithography (UVL)

Starting with a wafer that we want to pattern, which gets coated with a photoresist and patterned glass which get lighted. That causes some material to dissolve and some not.

• negative photoresist

• positive photoresist

—> often nice and flat, and uses one single crystal and how the lattice is orientated is indicated by the miler indices

negative photoresist

solubility in the developer decreases with the exposure

• example: SU-8 epoxy which is quite suitable for thick engraved material

positive photoresist

Solubility in developer increses with exposure dose.

• example: high-mw PMMA

spin-coating (resist layers)

The waver rotates and spin coating layer is deposited on the rotating waver.

—> higher speed means a thinner film (10 nm-100 microm)

other coatings: resist casting, spray coating and dry film lamination

the photomask

The part that lets through the light. This is most of the time glass, but it depends on the nm. Cronium absorbs a lot of light and therefore has a high optical density.

• line glass used for short wave lengths

• edge lengths most of the time 4 inch

note: most of the time cronium is made with laser/e-beam lithography.

different lithography techniques

Contact or UV proximity (UVL), X-ray lithography (XRL), electronbeam, digital light processing (DLP), thermal hot embossing and nanoprint lithography (NIL), laser direct writing (LDW), two photon polymerization (2pp)

2 pp laser lithography

layer by layer fashion. An ultrashort pulse laser beam is scanned through a photosenstivie resin. Due to high intensity in the focal point, 2 photons are absorbed and polymerization/ cross-linking happens.

digital light processing (DLP)

Based upon mirrors and how these are positioned to create an ‘on’ or ‘off’ idea. The Light cross-links the materials also in a layer by layer fashion.

nanoimprint lithography (NIL)

Thermal based: A thermal plastic resist is placed, that is heated end pressed at a temp above the Tg. Those are than cooled and seperated.

UV-based: A photopolymer resist is placed and UV-light combined with pressing happens. Than they are seperated

two working principles

• substractive: isotrpic wet ething, anisotropic Si wet etching, and dry/plasma etching. A high aspect ratio is created with Deep reactive ion etching (DRIE)

• additive: Build on the top of the wafer (electroplating, falvanoforming of metals on the wafer with electrically conductive layer, evaporation, sputtering or lift-off.

DRIE

is a very precise etching method mostly used for deep or narrow structures (micro-electro-mechanical systems)

steps:

1. plasma generation

2. ion bombardement (removing or etching the material)

3. anisotropic etching (etching directional matter)

4. stop layer deposited for etching

differences between etching, lift off, electroplating and stencil masking

etching removes material

lift off removes unwanted deposited material

electroplating deposits material by electrical reduction

stencil masking deposits material controlled with a mask

physical vapor depositioning (PVD)

the process of physically adding thin filmlayer. Particles land on a substrate.

There are different types: evaporation, laser, sputering, chemical vapor depositioning

e-beam evaporation

Happens in vacuum whereby source material is evaporized by the e-beam and hitted by electrons. Once it is vaporixed it forms a cloud and is guided with a magnetic field. Most of the time the cooling of the wafer is crucible.

• shutter prevents contamination

• anode+cathode help guide and accelerate

sputtering

a negative cathode with a target material gets striked by plasma and atoms are ejected.

bluk micrmachining vs surface micromachining

bulk: Involves etching deep into silicon wafer to creatae structures and is typically done on a whole wafer and etches through it.

surface: Structures are build up by growing or depositioning layers on the surface instead of etching. Thin films are often later etched. The sacrificial layer, isolation layer, structural layer and micromechanical layer.

laser micromachining

Is used for cutting, engraving or modifying materials (frilling/ milling and has F-theta lense)

steps:

1. focus of the laser beam

2. beam expansion and the focus

3. laser movement

4. material interaction

5. post-processing

(macroscopic) thermoplastic injection molding

A moving screw plays an actie part in this process. The screw is moved forwards and the liquid therefore can be injected. The mold itself can be opened and the material can be taken out of it.

• small parts weights and shot weights are what is injected in the parts

• micomold inserts are attached to an injection molding tool

• evacuation of the molding space

• A good homogenity of the molding material is need just like a precise dosing and metering and injection.

• The mold cavity is heated

• A low viscosity of molding material and a high injection pressure is needed

• play free guidance tool opening

Hot embossing

it is a bit like NIL, also starting with a plate, mold and subset plate. Those are heated and than pressed together. They stick to the starter plate.

• used for thermoplastic plats, films and sheets

• Important is to leave space between the plate-type micromold and the substrate

• mold space is getting evacuated

• polymer is heated in the mold and cooled down in the same variothermal process

• adhesion to the substrate is important

• sometimes removal of resudal layers or dicing is needed

micro pressure (thermos)forming

Polymer is softened but not liquified for this process, such as in micro blow molding and not such as in micro injection. The film is than pressured and formed. This is done with differential pressure. Hot embossing sometimes goes with a slightly flowing materials, micropressure however the temp is just Tg< depending on the crystallisation.

(macroscopic)blow molding

Looks a lot like glass molding, the parison that is hollow tubed is extruded in the hollow mold that surrounds it due to the steam of the air. The material is pushed in the mold and once it solidifies the mold opens again.

LIGA (lithography, galvanik, und abformung)

made out up of 3 methods:

1. lithography: the proces where a mask is placed to define a pattern mostly due to x-ray photolithography and due to resist eventually the structure is chemically altered.

2. electroplating: used to build a micrstructure, often after lithography and to add metal to the structure. Hereby an electric current runs through a solution and metal ions are deposited on the substrate (creating 3D structures)

3. Making a replica of the made structure for mass production.

adv: high precision, scalability and material versatility.

metrological methods

• styllus profilometry for surface and height with a needle that scans parts and is measured 13

• atomic force microscopy (AFM) for surface topography works the same as a stylus 13

• confocal laser scanning microscopy (CLSM), measures the vertical side walls. IS very precise and looks at the lateral direction. 23

• White light interferometry (WLI) mostly used for surface characterization and is a electron modality microscope 2

• scanning elecron microscope 23

1. tactile-contact- mechanical based

2. not contact based/ optical

3. scanning based

microfluidics+ adv

the science of systems that process or manipulate small amounts of fluids

adv:

• less weight

• less space requirements

• less material consumption

• less energy consumption

• smaller sample and waste volumes

• faster analysis/ synthesis

• increases spatial control

• reduced associated costs

Reynolds number

tells us something about the fluidic

• Re= inertial forces/ viscous forces

• you can hereby identicate laminar (minimal mixing due to layer by layer moving)or turbulent (easy mixing) flow

—> for mixing ofcourse micromixers can also be added. The inlet and outlet flows are the places wehre flow occurs.

Hagen-Poiseuille equation

Is for calculating the flow rate (Q)

Calculation is dependent on whether it is a rectangle or a circular system.

Though only to be used for small systems, because it does not take into account the shear stress. It shows for an incompressable fluid throug a pipe or a channel. In bigger systems those are seen and thus this formula cannot be used.

parallel channels

P= P2=P1

Q=Q1+Q2

R= (R1*R2)/(R1+R2)

series channels

Q1=Q2=Q

P=P1+P2

R=R1+2

inducing flow, different methods

• mechanical displacement pumps (syringe or peristaltic)

• pressure controllers

• micropumps

• capillary forces

• gravity

• centrigugal forces

• electroosmotic forces

maintaining a stable flow rate

• pressure to fluid in the microchannels

This pressure causes the fluid to be maintained. Creating a feedback loop.

• valves (membranes or ball valves) are influenced by external stimuli like pressure, magentism, electricity or temperature.

measuring flow on microscale

• a thermometer structure whebery time and distance is measured and thereby the flow can be calculated

• flow sensor/ anemometer

• Two measure points are taken

measuring pressure

can be done with a strain-gauge based micro pressure measurer. It measures the strain/ deformation.

mixing of microfluidics

• passive: Happens due to the geometries of the system and no external powers. (laminations, injection)

• active: Involves external forces er energy, pumps. (magnetohydrodynamic, acoustic, waves, thermal and high energy)

Staggered herrinbone mixer (SHM)

A specialised microfluidic mixer that is used in calcification risk. It makes complex fluid flow patterns to efficiently mix fluid in very small channels. Creating more interafce and making it more efficient.

gradient generators

• 1D chambers: used to create gradient profiles of a substance in a microfluidic devie, studying diffusion and concentration.

• 2D diffusion chambers: create an orthogonal gradients, channels overlap most of the time and they create these gradients in every direction. Every gradient is created in the perepndicular direction, showing more complex situations.

• T-junction: Two different fluids flow into the junction from seperate channels. One fluid enter the horizontally side and the other the vertical siede. Two fluids thereby mix and create a gradient.

• dilution network, ‘christmas tree’: A more complex dilution network. Which involves branching of the networks and flow thorugh smaller and smaller channels. Which is more suitable for complex systems.

microTAS

has everything for prepping, chem and biochem analysis and is integrated on a chip. (reaction and detection)

Lab on a chip LOC

is an extentsion of microTAS, with also data analysis, detection and manipulation.

PDMS

often used for microfabrication

adv:

• easy and cheap

• high flexibility

• high gas permeability

• high transparency+low autofluorescence

disadv:

• slow fabrication

• uptake+release small hydrophobic mol

• flexbility not suitable for high pressure applications

• permanent recovery after surface modifications

different types of microfluidic platforms

• minitaturized total chemical analysis system

• lab on a chip

• lab on a tube

• droplet-based microfluidics

• paper-based—> suitable low income

• digital

organ on a chip

mimics the key aspects of the human physiology (structure and function of human organs)

Instead of 2D structures they control the input and output of toxins.—> so it is more nature relevant

key aspects: being able to do experiments with cells from humans outside of the body, they contain microchannels, control microenvironments and allow real-time observations and can be powerful for physiology and disease models.

—> fill the gap between complex animal models and 2D models which causes it’s relevance

advantages of an organ on a chip model

• physiological relevance

• controlled microenvironment (compartilization)

• multi-organ integration (for system studies)

• reduced reliance on animal testing (faster, more ethical and the costs)

• predictivve power drug testing

• customization and personalized medicine

• high-throghput and scalable (automation, AI, multiple screening)

designing a organ on a chip steps

1. conceptualization + design

2. materials selection (dependent on functionality)

3. selection of biological elements

4. supporting life inside the organ on a chip

5. read outs

conceptualization+ design

1. Do you want to recreate one organ (or tissue) or multi-organs (several organs/ body on a chip—> focus on interaction between)

Simplifying where this can be done.

2. Is it a solid organ or a barrier? Solid organs are; bone, tumour, liver, pancreas, cartilage mostly 3D masses or embedded in the ECM. Barrier organs are; endothelial and epithelial cells (skin/gut), they regulate active transport of molecules and a membrane can seperate environments.

materials selection (dependent on functionality)

For the chose idea a microfabrication strategy should be determined just as the read-outs options and biocompatibility. PDMS is often used in these systems due to its transparency, elasticity( could be used as stretchable membrane or for mechanical tests), gas-permeability and biocompatibility.

But it can also absorb small molecules such as growth factors.

—> compromise between function, acces and fabrication facitilities.

After fabrication sterilization is often required

sterilization methods

• autoclaving (pressurised steam)

• ethanol

• UV

• surface treatments (also used for better biocompatibility or cell adhesion—>protein coatings or ecm

  → depends on the used material, for organoids or 3D spheroids we do not want attachment of cells and therefore we use pluronic acid often

selection of biological elements

two approaches

top-down: Using primary tissue (organ slices for example)or engineered tissue in the ooc. Those are very important for personalized medicine and specific cases. The culturing of the cells and isolation of those is done, outside of the chip.

bottom-up: Isolated cells such as primary, immortalized or stem cells are directly cultured on a chip. The microenvironment is crated for the specific tissue.

options for cells in bottom-up

• primary cells: retain full function which is seen in vivo but are difficult to isolate, batch-to-batch variables a lot. (dedifferentiation is sometimes needed)

• immortalized cells: easy to grow and robust/reproducible. However they are modified to promote themself forever.

• IPCS: derived with the minimal invasive procedures—> often very good option

functional time window

period that is required to develop and maintain physiological conditions for cells.

supporting life inside organ on a chip

• selection of cell culture medium depending on how many types of cells and may require optimal medium to maintain also the phenotype

• perfusion/ delivery of the medium (via microchannel)

  —>could be by convective flow, driven by pumps or gravity

  • one-pass perfusion flow: syringe pumps and stable supply of fresh nutrients and downstream inter-tisue. Most of time with reservoir spent medium.

  • recirculary flow:peristaltic pump and recirculting signalling molecules with accumulation of waste products

—> important to focus on controlling microenvironment

Damköhler number

tells why change is seen, due to velocity or by things in the chips that are biologically

peclet number

tells whether transport dominated by advection or diffusion

ALI Air liquid interface culture

used for several purpouses:

• cell differentiation+ maturation

• encouraging cilia formation, mucus production

it is a way of supporting the life inside the device, whereby cells are in the middle and one side with air and the other with medium.

read outs

• in situ/real-time: optical imaging, barrier function assays, integrated sensors, molecular and functional read outs

• end popint/ after experiment with harvesting cells fixation or media collection: gene expresion, cytokine/protein analysis, structural and morphological analysis, metabolics and lipidomics and functional assays.

mechanical stimulation that cells normally experience

• active: direct consequences of organs, tissues that are used to constant stretching (mimicked with vacuum chambers, flexible membranes or mechanical actuators)

• passive: indirect experience. Such as endotheial cells in connective tissue cells. This can be done with fluid perfusion in microchannels.

stress/strain—> mechanotransduction—>proliferation,migration,phenotype and differentiation

limitation organ on a chip

• variability (batch to batch)

• donor to donor variability in genetics for example

• line to line variability (patient heterogenity)

• standardization—>not easy and done yet

• data+ validation gathering

different methods for synthetic porous cell culture membranes

ion tracking, phase speration, solvent casting/particulate leaching, electrospinning of polymers, melt electrowriting, PDMS molding over micropillar (pores), photolithography and etching (depending on membranes)

ion track etching

A technique to creat well-defined narrow pores or channels. The combination of high energy-ion radiation with chemical etching. Membran most of the time s-10 microm thick.

the ions make the holes and the etching deepens the holes.

→ mostly produce cylindrical and perpendicular to the membrane structures which is good for visualization.

phase seperation PSmicroM

can happen 2 ways

• NIPS with a polymer solution and solvent

• TIPS with temperature

This creates porous or patterned structures

PDMS molding over micropillars

basically pressure while on micropillars will create a porous structure

• high costs

• membrane could be stretchable (usable for biomimetic cultures:collagen membrane (type 1),collagen IC-laminin basement like membranes, surface-immobalized lipid by layers)

micropatterning of the substrates, different types of patterns

patterns organize and pattern the cels but also influence the cells faith.

• subcellular (near cell scale or size of multiple cells)

• discrete (high-low, adhesive-non-adhesive, soft-stiff) or smoothened

• gradients either discrete or continious—> on feature size and density

• synthetic, bioinspired/mimter or nature-derived

how to do topographical screening?

• Topochip an be used, which is topographical library for screening cell-surface interactions.

• galapagos chip: cell-adhesive library for scree cell-surface interactions

• pocket lithography—> choosing a small piece to do lithography on

chemical ways of patterning

• photopatterning

• micro contact printing

• dip pen nanolithography (DPN)

DPN dip pen nanolithography

A pen in the form of an AFM tip that dips into a biomolecule ink and depostits patterns of biomolecules on a substrate in the form of a self-assembled monolayers (SAMs) An increased stiffness causes a higher rigidy.

different curvature engineering processes

mechanical micromachining, lithography, isotropic wet edging, lithography+thermal reflow of photoresist structures, laser micromacining/ ablation. Microtunable mold derived techniques, microthermoforming of thin films, stereolithography and 2 pp laser lithography.

soft lithography

using elastomer stamps, conformable photomasks and molds (soft and moldable ones)

different types of molding +description

• replica molding (REM): a mold of PDMS is given and a prepolymer is placed on that cured and peeled of.

• microTM, a prepolymer is placed on support. That is cured and the mold is removed and placed on the support. Residual film is left.

• MIMIC, a PDMS mold is placed on support and drops of a prepolymer is placed at one end Channels are filed by capillary action and those are cured and the mold is removed.

• SAMIM, starts with wetting the liquid (solvent) and PDMS mold. That is placed on a support (film on top of support). The solvent gets evaporated and the mold gets removed, leaving the support, residual layer and softened polymer that is changed due to the mold.

• microcontact printing (microCP) is as well inking as stamping. A master is created with lithography and used as a stamp which could be inked. Those cacn habe cehm reaction from transfer of the stamp on the glass. These create a highly spatial controlled space/arrangement of cells. Sometimes pre-conditioning is needed.

A cell culture

growing cells in a controlled environment, outside of their native environment (culture dish), those are controlled in every sense.

Culture environment needs:

• nutrients

• gases and gas exchange

• physiological environment

• sterile environments

• adhesion for adherent cells

2D cultures

Culturing cells on flat substrates such as tissue culture plastics without or with ecm coatings → monolayers

adv of 2D culture

• easy and cost-effective

• standardization and established protocols

• comparitive studies

• easy access to cells for analysis or manipulation

disadv of 2D culture

• cell-cell attachments only in corners

• adhesions to matrix only on one side

• forced apical-bassal polarity due to anisotropic contact

• continious layer of the ecm-→ this is not in reality

• unconstrained spreading and migration

• high stiffness substrate (sandwhich method)

• absence of soluble gradient

3D culture

Culturing cells in 3D environment mimicking the 3D in vivo tissue environment

3D advantages

• our body is not 2D so more physiological relevance

• aggregation of cells, matrix, gel or scaffold

• cell-cell contact diff layers

• 2D cell- matrix interactions (adhesion)

• no forced polarity

• proper mech cues

• soluble gradient→ takes time for molecules and signal to travel

• tunable stiffness

• miniaturization possibility

• compatible to downstream analysis

disadv 3D cultures

• high costs but can be made cost-effective

• time consuming

• more complex to analyse due to more layers

• standardization still getting worked upon

3D cultures types/methods

scaffold free:

• self assembly into spheroids:

  • liquid overlay

  • rotary cultures

  • spinner flask

  • hanging drop culture

  • culture in microwells

• embryoid bodies

• organoids

  • matrigel dome culture

  • microwell clture

  • organ on a chip

scaffold based:

• cells seed onto scaffld

• cell laden hydrogels/ bioprinted structures

cells seed onto scaffold

Scaffold provides structural support and mimics the exm, which allows cell attachment, proliferation and tissue formation. Allows nutrients to diffuse.

creates porous, biocompatible, tuned mechanical properties and designed architecture (meso, micro and nano)

cell laden hydrogels/ bioprinted constructs

Are 3D in vitro models weherby living cells are encapsulated or embedded within hydrogel matrix.

Hydrogels are 3D networks of hydrphilic polymer that are formed by crosslinking of the polymer (retain lot of water), they have molecular pores.

cells seeded on microparticles

4 main ideas for why using this

A) cell expansion, looking at giving cues or temporary support

B) direcct filling for creating structural support and anchoring points to the injected cells.

C) 3D tissue/ disease modelling; looking at self assembly of cells with different cues

D) bioinks, need control of rheological properties and mechanical stability

self assembly into spheroids

cells are in a non-adherent environment and come into contact with each-other. They adhere via cahersin forming clusters. The cytoskeletal forces pull the cells thightly together.

• layered zones are created→diffusion gradient

• outer to inner:proliferating, quiescent, necrotic

Necrotic should be prevented, by making spheroid size withing the diffusion limit or adding microparticles to help create porous environment (making structure less dense). Or vascularization of the spheroid (making a bath of endothelial cells trying to get vascularization)

liquid overlay

Cells are seeded into regular flat-bottom or U-bottom wells which have non-adhesive surfaces. they can’t attach to the surface so they aggregate due to gravity and cell-cell interactions.

adv: simple and inexpensive and works in standard lab equipment.

disadv: less control of the spheroid size/uniformity, cells might form irregular aggregates if density is not optimal and limited scalability for high- throughput needs.

rotary cultures

A microgravity was created with draggging forces and is quite a neat method. A low shear stress was seen and cells were not damaged. It has a slow rotating horizontal cylindrical vessel, filled with medium+cells. The cells constant are in a free fall state where the cells are allowed to float and interact naturally.

adv: low shear stress and fgood uniform spheroid formatio at scale.

spinner flask

A stir bar or impeller is used to agitate the culture medium. Agitation keeps the cells in suspension and prevents them from settlng on the bottom of the flask. The cell-cell interaction is imp and the stirring speed should therefore be optimized.

adv: simple set-up, scaling up, allows diffusion by mixing

hanging drop culture

placing small droplets of cell suspension on the inverted surface. The gravity pulls the cells to the bottom of the droplets where those aggregate and form spheroids. The liquid is often in a well to prevent evaporation of the droplet.

adv: cost-effective, simple, no need for specialized equipment, high reproducibility and control over spheroid size.

disadv: limited scalability, manual handling, build up nutrients/waste in drop and influence on long-term.

culture in microwells

Using microfabricated microwells with non-adherent materials. Cells are trapped and lead them to self-aggregation and form a uniform spheroid.

Controlled→ high univormity

adv: High control spheroid size/shape, scalable and high throughput, allows formation of hundreds to thousand of spehoirds in parallel. the are reproducible and downstream analysis is possible.

disadv: requires specialized plates, fabrication techniques, slightly more complex than liquid overlay is costlier than basic liquid overlay.

embryoid bodies

A 3D cluster of pluripotent stem cells like embryonic stem cells or induced pluripotent stem cells. Thoseorganize and differentiate into varioius cell types when cultured. They are used to study early development processes, observer differntiation into 3 germlayers or test toxicity/ kick-off directed differentiation processes.

→ aggregate just like as in embryogenesis.

organoids

3D self organizing structures derived from stem cells (pluripotent or adult) they mimic the architecture and function of real organs in vitro.

→ miniature/simplified versions of organs.

• stem cell derived

• 3D structure

• cellular diversity

• tissue/cell organization

• functional properties—> functional tissue pieces

top down TE

Cells are seeded onto a pre-formed scaffold, attach, grow, produce matrix and form a tissue

adv:

• benefit from the scaffold

• various fabrication techniques ( tuned chem compo, porosity and mech prop)

• suitable large tissue constructs

disadv:

• uniform cell distribution is a challenge

• limited spatial control

• lack cell guided tissue formation

• vascularization= requirement

• high cell conc difficult→no control of the cells

• scaffold guided tissue form

bottom-up TE

building tissues by creating small functional tissue building blocks which assemble modularly into larger more complex structures.

→ can be made from cells, aggregation cells, microtissues with or withou biomaterial/molecules. Mimics the natural formation.

adv:

• precise control of environment+better mimicry of native tissue

• cell guided tissue formation and remodelling

• control spatial positioning

• enabling vascularization due to including vascular building blocks

• allow large scale prodution microtissues to larger tissues

  →no one actually proved these advantages