Cell seeding on scaffolds
A concentrated drop of cell suspension is pipetted onto a damp scaffold and wicked into its pores; the cells settle and attach before medium is added. Simple and widely used, with efficiencies typically between about a quarter and two thirds and a top-heavy distribution.
Our ruleSimulations prepare you for the bench but don't replace hands-on practice.
Static seeding
A concentrated drop of cell suspension is pipetted onto a damp scaffold and wicked into its pores; the cells settle and attach before medium is added. Simple and widely used, with efficiencies typically between about a quarter and two thirds and a top-heavy distribution.
How it differs from the standard method
- The seeding volume should match the pore volume; larger drops run off.
- Leave 1–3 h in a humid incubator before flooding the well.
- Seeding both faces (flipping the scaffold) helps thicker discs.
1Getting cells into a sponge
A tissue-engineering scaffold is a porous solid, often more than 90% void, that cells are meant to colonise. Seeding is the step that puts them there, and it decides much of what follows: how many cells the construct starts with, whether they reach its interior, and how evenly they are spread.
Drop seeding
A small, concentrated drop is pipetted onto a damp scaffold, which wicks it into its pores. Simple and cheap, but limited by the scaffold's pore volume and always densest at the face the drop went on.
Spinner, orbital or perfusion
The suspension is moved past or through the scaffold so that cells meet the pore walls throughout. Oscillating perfusion, which pushes the suspension back and forth through a press-fitted scaffold, gives the highest and most even seeding in thick scaffolds.
Live/dead in 3D
Calcein-AM marks cells with active esterases and an intact membrane green; ethidium homodimer enters only damaged cells and stains their nuclei red. In a thick construct, what matters is where you look.
2What sets the efficiency
In static seeding the pore volume is the first limit: a 5 mm by 2 mm disc at 90% porosity holds about 35 µL, and any extra volume runs down its sides. The scaffold must also take the liquid in, which a dry, hydrophobic polyester does poorly and a scaffold already full of medium cannot do at all.
3Measuring it honestly
The direct measurement is to move each scaffold to a fresh well, digest it and quantify its DNA against a standard curve made from the same cells. The tempting shortcut is to count the cells left in the medium and subtract them from the number seeded, but that books every cell stuck to the plastic or the tubing, and every cell that died and lysed, as seeded.
4Why the core dies
Once seeded, a construct in static culture is fed only by diffusion. Cells use oxygen faster than it can diffuse far, so beyond a critical depth of a millimetre or so (less as cells multiply) the interior becomes anoxic and dies over days.
Key terms
Practise these as flashcards →Cells attached in the scaffold after seeding divided by cells seeded, best measured by DNA in the scaffold.
The void fraction of the scaffold's volume; it sets how much liquid the scaffold holds.
Whether pores open into each other; isolated pores cannot be reached by cells or medium.
The volume of liquid a scaffold can hold, porosity times its bulk volume.
Pumping the cell suspension back and forth through a scaffold sealed in a chamber, so all the liquid passes through the pores.
The drag of flowing medium on cells lining the pores; a few mPa is tolerated, tens of mPa start to detach newly attached cells.
A non-fluorescent, membrane-permeant ester that live cells cleave into green calcein and retain.
A red DNA dye excluded by intact membranes, so it marks dead or damaged cells.
The depth at which oxygen consumed by the cells above uses up what diffuses in; cells below it become anoxic.
When to use it
- Starting any 3D tissue construct on a porous scaffold, where the starting cell number and distribution need to be known and reproducible.
- Comparing scaffold designs (pore size, porosity, coating) by how well they take up and hold cells.
- Checking, with a live/dead stain through the depth, whether a construct's culture conditions keep its interior alive.
Compared with alternatives
Where pore size, porosity and interconnectivity are decided; fix the scaffold there if cells cannot get in.
Perfusion culture after seeding keeps thick constructs alive where static culture lets the core die.
Encapsulating cells in a gel puts them evenly through the volume from the start, at the cost of a matrix they must degrade to spread.
Plate-based viability assays are faster for monolayers but give one number per well and no depth information.
The optics behind the live/dead stacks, including how deep a scattering sample can be imaged.
Sub-techniques
Choosing one here switches the whole page, including the protocol and the lab, to it.
A concentrated drop of cell suspension is pipetted onto a damp scaffold and wicked into its pores; the cells settle and attach before medium is added. Simple and widely used, with efficiencies typically between about a quarter and two thirds and a top-heavy distribution.
- The seeding volume should match the pore volume; larger drops run off.
- Leave 1–3 h in a humid incubator before flooding the well.
- Seeding both faces (flipping the scaffold) helps thicker discs.