[1] Disposable, sterilized cell strainers:BD #352350 70um

Reusable cell strainers: Dual Mfg #US3-200S 74um

(Brass frame/stainless steel mesh; #200 mesh/74um; 3" diameter x 1" depth)

Disposable BD strainers fit over standard 50mL conical tubes

Reusable Dual Mfg strainers work best with 95mm diameter culture plates; the lip on the strainer frame sits on top of the 95mm plate perfectly, while 100mm plates are a bit too wide, causing the strainer to sit entirely on the bottom of the plate.

Dual Mfg strainers are autoclavable, although we have not found it necessary to autoclave them every time. Washing well with detergent and hot water, followed by thoroughly spraying with 70-75% ethanol is sufficient. Make sure you allow the ethanol to dry (either air-dry in the hood, or use a heat gun) before use. Furthermore, make sure you wet the bottom of the strainer immediately before use with sterile media, to facilitate flow-through.

Do NOT expose cell strainers to direct fire- the solder used to seal the mesh to the frame will melt. Likewise, if using a heat gun, do not use too high a temperature setting (<300F).

[2] These cannulas have a large beveled tip, which aids flow rate, but also requires some experience to properly insert into the portal vein. A viable alternative is to use fine-bevel Teflon-sheathed cannulas.

[3] See formulation sheet for ingredients. The HBSS is formulated based on Invitrogen #14175, with the addition of 0.5mM EGTA and 25mM HEPES, sufficient NaOH to bring the pH to 7.4@37C. NO oxygenation of any buffers or media is necessary.

[4] We have had success with Mediatech/Cellgro DMEM-Low #10-014-CM, which contains 200mg/L CaCl2 (~1.8mM).

[5] See formulation sheet for ingredients. This contains 10% FBS, which helps to quench collagenase activity; the fibrinogens in serum may also aid in cell attachment, even in the presence of a collagen coating. If you wish to conserve media, perform the initial wash and final resuspension in isolation medium, and the intermediary two washes with serum-free medium (i.e. 50/50 DMEM-High/F-12).

DMEM-High: Mediatech/Cellgro #10-013-CV

F-12: Hyclone #SH30026.01

[6] The type and batch of collagenase you choose for isolation is absolutely critical. We and others have found that many batches of Type I collagenase, which is the most common form used in the literature for primary hepatocyte isolation, is far too harsh, and tends to damage cells and/or cleave receptors, compromising viability and function. We highly recommend a Type IV collagenase, often used for beta cell isolation, as this type contains ~10x less tryptic activity.

Worthington is strongly recommended as a source for collagenase; their customer service and quality control are exceptional, and their collagenase sampling program allows you to test batches of collagenase before committing to a specific lot.

[7] There are various standards used for assessing collagenase activity; most collagenase powders (including those from Worthington) contain other enzymatic activity which have been found to be important to digestion. As a result, collagenase selection is not an exact science, and batches must be tested. To ensure consistency, it is recommended that collagenase be quantified solely on the basis of its actual action of collagen digesting activity. In other words, 100 CDU/mL refers to the collagenase activity, exclusive of other enzymatic (e.g. tryptic) activity- hence the importance of selecting the right type and batch of collagenase.

We have found 100 CDU/mL to be an optimal concentration of collagenase; to adjust for liver size/animal age, simply vary the total volume and/or flow rate. For example, you may use 100 CDU/mL even for a rat, so long as you scale the flow rate and total volume as needed.

[8] Type I Collagen from rat tail: BD #354236. This comes in 100mg lots of varying concentration (generally ~3.5mg/mL) dissolved in 0.02N acetic acid. The concentration of collagen you use to coat your wells can have dramatic effects on the attachment, growth, and performance of your cells. According to BD, the concentration of collagen they use for coating is between 5-8ug/cm2; cm2 refers to the surface area of the bottom of the well- check the manufacturer's specifications, as surface area may not scale linearly with well size. We recommend 5ug/cm2 as a general rule. Plating by this method is not only more consistent than the standard "pipette 1mg/mL collagen in, remove excess, repeat for next well" method, but also much more economical.

[9] For actual coating, add the desired volume of collagen/0.02N acetic acid, shake/tap the plate to distribute the collagen evenly, and let dry under UV for 1.5-2 hours, or overnight. Rinse with PBS before use. You may use a repeater pipette to speed the process up- just make sure that you allow the plates to have sufficient UV exposure if you are using non-sterile repeater tips. For coating, simply add the required quantity of collagen in enough volume of 0.02N AcAc to easily coat the bottom of the dish, but not so much as to significantly coat the sidewalls (to minimize waste and evaporation time). For example, ~100-150uL of total volume/well for a 12-well plate, and 50-75uL/well for a 24-well plate.

Collagen coated plates may be sealed with parafilm and stored at 4C for months; it is generally recommended, though, that you use plates within two weeks.

[10] See notes in [1] for cleaning/sanitizing reusable cell strainers.