raft™ 3d cell culture kit protocol

RAFT™ 3D Cell Culture Kit Protocol

BioResearch

2 BioResearch – RAFT™ Kit Protocol

PreparationBefore you start, read the Notes, Hints and Tips on page 7. Then prepare the following items:

From the RAFT™ Reagent Kit (catalog numbers 016-0R94 and 016-0R95)

– Reagent bottles (chilled to 2–8°C) – Collagen, volume ≥100 mL for 016-0R94 and ≥25 mL for

016-0R95 – 10x MEM, volume ≥15 mL for both 016-0R94 and 016-0R95 – Neutralizing Solution, volume ≥10mL for 016-0R94 and

016-0R95 – Mixing vessel (chilled to 2–8°C)

When using a RAFT™ Plate Kit with 96-well plates (catalog number 016-0R92, and 016-0R96)

– 1 RAFT™ Absorber Plate: 96-well – 1 Greiner Culture Plate (clear), included with catalog number

016-0R92; alternatively, RAFT™ 96-well Absorbers can be used with black-walled Cell Culture Plates (96 well, µclear®, black, Greiner, PN: 655090)

When using a RAFT™ Absorbers for 24-well plates (catalog number 016-1R32 and 016-1R34)

– Have the correct number of absorbers on hand in their sterile packaging

When using RAFT™ Insert Absorbers for 24-well inserts (catalog number 016-1R33 and 16-0R97)

– Have the correct number of absorbers on hand in their sterile packaging

Also required but not provided: – Cells and suitable culture medium

(medium required even if no cells are used) – General lab tools, e.g., pipettes, sterile tips, serological pipettes,

chilled reservoirs – Ice in a container suitable for use in a laminar flow hood – If using the RAFT™ Absorbers for 24-well plates, an appropriate

24-well culture plate is required (not provided) – If using cell culture inserts, take the appropriate number of

RAFT™-compatible inserts out of their packaging and place them into an appropriate 24-well plate (neither provided).

NOTE:

24-well plate

To ensure optimum 3D cell culture production, the use of 24-well plates (tissue culture treated) from Corning, Greiner, Millipore and Nunc is recommended. 24-well plates (tis-sue culture treated) from other manufacturers may be used, however these have not been tested and may result in inconsistent cultures.

Inserts

To ensure optimum 3D cell culture production, it is recommended to use 6.5mm (in di-ameter) inserts from Greiner or Millipore, together with the relevant 24-well cell culture plates from these manufacturers - see below for full details. Inserts from other manufac-turers may be used; however these have not been tested and may result in inconsistent cultures.

– Millipore PIHT12R48 Millicell 0.4 µm translucent PET hanging insert – Millipore PIRP12R48 Millicell 1.0 µm transparent PET hanging insert – Millipore PIEP12R48 Millicell 8.0 µm translucent PET hanging insert – Greiner 662640 Thincert 0.4 µm translucent PET hanging insert – Greiner 662641 Thincert 0.4 µm transparent PET hanging insert – Greiner 662610 Thincert 1.0 µm transparent PET hanging insert – Greiner 662631 Thincert 3.0 µm translucent PET hanging insert – Greiner 662630 Thincert 3.0 µm transparent PET hanging insert – Greiner 662638 Thincert 8.0 µm translucent PET hanging insert

This protocol describes in detail how to produce RAFT™ 3D Cell Cultures in 96-well plates, 24-well plates and 24-well inserts. It is for use with all the RAFT™ Kits available through Lonza BioResearch. See our web site and price lists for further details.

A video of process animation and additional product information such as MSDS sheets and technical notes can be found at: www.lonza.com/raft

RAFT™ 3D Cell Culture Kit

RAFT™ Kit Protocol – BioResearch 3

ProtocolThe volumes in the protocol below will produce one fully populated 96-well plate, 24-well plate or 24-well inserts.

Note: For fewer cultures than in the table below please refer to the Reagent Volume Guide on pages 8–10.

Plate Formats Number of Wells Volumes (mL)

10x MEM 2 mg/mL CollagenNeutralizing

SolutionCell Stock Solution

Cell Seeded Collagen Solution

n x y z c d

96-well Plate 96 2.800 22.4 1.624 1.200 0.240

24-well Plate 24 3.500 28.2 2.042 1.500 1.300

24-well Plate Culture Insert 24 0.780 6.2 0.452 0.328 0.240

Full protocol available for download at www.lonza.com/raft

Process Overview The process has five simple steps:

1Prepare

Collagen Solution

2Prepare

Cell Stock Solution

3Mix

Collagen Solution and Cell Stock Solution

4Plate and Form

Hydrogel (15 mins)

5Make

RAFT™ 3D Cell Culture (15 mins)


1. Prepare Collagen SolutionCarry out the process below with the reagents and mixing vessel on ice. ➊1.1 Add the volume x of 10x MEM into the supplied mixing vessel

(Fig 1).1.2 Slowly add the volume y of the collagen solution into the

mixing vessel. ➋ ➌

1.3 Trying not to introduce bubbles, swirl the solution carefully around the mixing vessel to mix (Fig 2). A correctly mixed solution will have a homogenous color. ➍

1.4 Add the volume z of neutralizing solution dropwise around the surface of the mixture using a P1000 or P200 pipet. Swirl the solution gently to minimize the introduction of bubbles (Fig 2). ➋ ➍

1.5 A correctly mixed solution will have a homogenous color. 1.6 Keep the prepared collagen solution chilled until required.

2. Prepare the Cell Stock SolutionAn appropriate cell culture medium must be prepared even if cells are not being used in the collagen gel. Harvest cells according to best cell culture practices. ➒ Avoid delays during cell harvest or between cell harvest and set-up of RAFT™ Cultures.

The table below gives examples of cell densities.

Resuspend cells in culture medium at the appropriate cell concentration. A minimum volume c of cell stock solution is required for the entire well plate.

Cell Concentration in the Cell Stock Solution (cells/mL)

Number of Cells per Well 96-well Plate or 24-well Insert Plate 24-well Plate

1,000 9.92E+04 1.83E+04

5,000 4.96E+05 9.16E+04

15,000 1.49E+06 2.75E+05

20,000 1.98E+06 3.66E+05

25,000 2.48E+06 4.58E+05

30,000 2.98E+06 5.49E+05

40,000 3.97E+06 7.33E+05

50,000 4.96E+06 9.16E+05

75,000 7.44E+06 1.37E+06

100,000 9.92E+06 1.83E+06

0 Culture medium only Culture medium only

Figure 1.

● See page 7 for “RAFT™ Process Tips and Hints”


3. Mix Collagen Solution and Cell Stock Solution (or medium)3.1 Slowly add the volume c of the cell stock solution from

step 2 (or, if no cells are to be used, medium only) into the collagen solution from step 1.6. ➊

3.2 Trying not to introduce bubbles, swirl carefully around the mixing vessel to mix the solutions together (Fig 2). Keep the cell-seeded collagen solution chilled until required. ➋ ➍

4. Plate and Form the Hydrogel4.1 Either transfer the solution from step 3.2 into a chilled

multichannel pipette reservoir or dispense it directly from the mixing vessel. Keeping the reservoir/mixing vessel on ice during this step will help delay gelling.

4.2 Dispense the volume d of the cell seeded collagen solution into each well of the 96-well culture plate (either clear or black-walled plate) or in a RAFT™-compatible 24-well plate or 24-well insert and replace the plate lid. ➌ ➑

4.3 Put the culture plate into a cell culture incubator set at 37°C (Fig 3). Do not stack plates on top of each other. Close the incubator and leave the culture plate for 15 minutes to form a hydrogel.

NOTE: When using inserts, it is particularly important to follow the guidance in hints ➌ and ➑. Use a single-channel pipette to ensure dispensing is centered in the inserts. It also may be helpful to stabilize the pipette with the other hand to ensure that it does not touch the side of the insert.

Figure 2.

Figure 3.



5. Make the RAFT™ 3D Cell Culture5.1 Retrieve the culture plate from the incubator and return it to

the laminar flow hood and then remove the plate lid.5.2 Place the RAFT™ Absorbers on top of the hydrogel. a If making RAFT™ Cultures in a 96-well plate, remove the

RAFT™ 96-well Absorber Plate from its packaging. Align the absorbers over the culture plate wells and confidently lower the RAFT™ Absorber Plate vertically without tilting and without sideways movement (Fig 4).

b If making RAFT™ Cultures in a 24-well plate or a 24-well insert plate, remove the appropriate number of absorbers from the absorber package by grasping each absorber by the wider end and then placing one on top of each hydrogel so that the narrower end is in contact with the hydrogel. You should not put any pressure on the hydrogel; merely drop the absorber on top of it. Do not put any pressure onto the absorbers. The process will begin under just the weight of the absorber.

5.3 Leave for 15 minutes at room temperature in a laminar flow hood.

5.4 Remove the absorbers from the culture plate. a If making RAFT™ 3D Cultures in a 96-well plate, holding

the culture plate down with one hand, gently remove the RAFT™ 96-well Absorber Plate from the culture plate (Fig 5). The RAFT™ Absorber plate can now be discarded.

b If making RAFT™ Cultures in a 24-well plate or 24-well insert plate, remove each absorber gently in turn; they may now be discarded. You may need to hold down the culture plate or the insert while removing its absorber.

5.5 On top of each RAFT™ 3D Cell Culture immediately add culture medium (e.g., 100 µL for a 96-well plate, 500 µL for a 24-well plate and, for an insert plate, add 100 µL into the insert and 500 µL outside the insert to the well bottom). If cells are being seeded on top of the RAFT™ 3D Cell Culture then add cells in medium.

5.6 Replace the culture plate lid. The RAFT™ Cultures are now ready for further incubation or analysis.

Figure 4.

Figure 5.



RAFT™ Process Tips and Hints➊ In common with handling any extracellular matrix protein solution,

it is very important to keep the solution chilled until gelling is required. Gelling (collagen fibrillogenesis) will commence on addition of the neutralizing solution; however the gelling process can be delayed by keeping the collagen mixture on ice.

➋ Because there are viscous and dense solutions in the kit, please aspirate and dispense each reagent slowly to avoid creating bubbles in the gel mix. Bubbles, once created, are difficult to remove from the solution and the subsequent culture and can interfere with analysis. If bubbles are evident it can help to leave the gel mix on ice for 10–30 minutes to allow some of the bubbles to dissipate prior to making the RAFT™ Culture.

➌ To improve the accuracy and precision of pipetting of the collagen solution and to reduce risk of introducing bubbles, it is advised to take a greater volume than required into a pipette and dispense it by reverse pipetting, e.g. for 22.4 mL solution aspirate 24.4 mL and dispense down to the 2 mL graduation of a 25 mL serological pipette.

➍ A homogenously mixed solution can be achieved more quickly by swirling clockwise and anti-clockwise in an alternating fashion. Using vessels (e.g., 50 mL tubes) other than the supplied mixing vessels may result in excess bubble formation. For small numbers of cultures it is acceptable to mix the contents in a sterile 15 mL centrifuge tube by gentle inversion.

➎ This protocol has been optimized for making a single complete plate at a time (whether 96-well, 24-well, or 24-well with inserts). The RAFT™ Standard Reagent Kit contains sufficient volume to use this protocol four times for 96-well plates, three times for 24-well plates, and for up to 384 cultures in 24-well plates with inserts. Modifying the protocol by increasing or decreasing collagen volume to change the resulting cultures is part of the versatility of the RAFT™ System. Modifications to the protocol will change the number of cultures that can be made successfully using a single kit.

➏ A volume guide has been provided for use when there is a requirement to make less than an entire plate at once. There is wastage of solutions in each receptacle and in each pipetting step and this should be considered when partially populating plates. An estimate of this wastage is included in the volumes stated in the guide.

➐ Cells being moved from a 2D into a 3D environment such as a RAFT™ Culture often require at least overnight culture to acclimatize. However, this is cell dependent and may have to be investigated as part of the experiment. For example, with fibroblasts, a morphology change is seen over the first day or two from round to an elongated form that is seen in tissues.

➑ Pipette into the centre of each well or insert rather than on the side to reduce the risk of collagen sticking to the sides of the absorbers. Finish the dispense below the liquid rather than on top of it to reduce bubbles and splattering.

➒ The RAFT™ Cell Culture System is based on rat tail collagen type I. Therefore, harvesting of cells with collagenase prior to setting up RAFT™ Cultures is not recommended. Traces of collagenase can interfere with collagen scaffold formation.


Reagent Volume Guide For various numbers of wells in a 96-well Plate (all volumes are in mL)

96-well Plate 10X MEM Collagen Neutralizing Solution Cells or Medium Total

x y z c

1 0.12 0.96 0.070 0.050 1.2

2 0.15 1.2 0.087 0.063 1.5

3 0.17 1.4 0.099 0.071 1.7

4 0.20 1.6 0.116 0.084 2.0

5 0.22 1.8 0.128 0.092 2.2

6 0.24 1.9 0.139 0.101 2.4

7 0.27 2.2 0.157 0.113 2.7

8 0.29 2.3 0.168 0.122 2.9

9 0.32 2.6 0.186 0.134 3.2

10 0.34 2.7 0.197 0.143 3.4

11 0.36 2.9 0.209 0.151 3.6

12 0.39 3.1 0.226 0.164 3.9

13 0.51 4.1 0.296 0.214 5.1

14 0.54 4.3 0.313 0.227 5.4

15 0.56 4.5 0.325 0.235 5.6

16 0.58 4.6 0.336 0.244 5.8

17 0.61 4.9 0.354 0.256 6.1

18 0.63 5.0 0.365 0.265 6.3

19 0.66 5.3 0.383 0.277 6.6

20 0.68 5.4 0.394 0.286 6.8

21 0.70 5.6 0.406 0.294 7.0

22 0.73 5.8 0.423 0.307 7.3

23 0.75 6.0 0.435 0.315 7.5

24 0.78 6.2 0.452 0.328 7.8

25 0.80 6.4 0.464 0.336 8.0

26 0.82 6.6 0.476 0.344 8.2

27 0.85 6.8 0.493 0.357 8.5

28 0.87 7.0 0.505 0.365 8.7

29 0.90 7.2 0.522 0.378 9.0

30 0.92 7.4 0.534 0.386 9.2

31 1.0 8.0 0.580 0.420 10

32-35 1.1 8.8 0.638 0.462 11

36-39 1.2 9.6 0.696 0.504 12

40-43 1.3 10.4 0.754 0.546 13

44-47 1.4 11.2 0.812 0.588 14

48-50 1.5 12.0 0.870 0.630 15

51-52 1.6 12.8 0.928 0.672 16

53-56 1.7 13.6 0.986 0.714 17

57-60 1.8 14.4 1.044 0.756 18

61-64 1.9 15.2 1.102 0.798 19

65-68 2.0 16.0 1.160 0.840 20

69 2.1 16.8 1.218 0.882 21

70-72 2.2 17.6 1.276 0.924 22

73-77 2.3 18.4 1.334 0.966 23

78-81 2.4 19.2 1.392 1.008 24

82-85 2.5 20.0 1.450 1.050 25

86-89 2.6 20.8 1.508 1.092 26

90-93 2.7 21.6 1.566 1.134 27

94-96 2.8 22.4 1.624 1.200 28


Reagent Volume Guide For various numbers of wells in a 24-well Plate (all volumes are in mL)

24-well Plate 10X MEM Collagen Neutralizing Solution Cells or Medium Total

x y z c

1 0.33 2.6 0.191 0.139 3.3

2 0.46 3.7 0.267 0.193 4.6

3 0.59 4.7 0.342 0.248 5.9

4 0.72 5.8 0.418 0.302 7.2

5 0.85 6.8 0.493 0.357 8.5

6 0.98 7.8 0.568 0.412 9.8

7 1.1 8.8 0.638 0.462 11

8 1.2 9.6 0.696 0.504 12

9 1.5 12.0 0.870 0.630 15

10 1.6 12.8 0.928 0.672 16

11 1.7 13.6 0.986 0.714 17

12 1.9 15.2 1.102 0.798 19

13 2.0 16.0 1.160 0.840 20

14 2.1 16.8 1.218 0.882 21

15 2.3 18.4 1.334 0.966 23

16 2.4 19.2 1.392 1.0 24

17 2.6 20.8 1.508 1.1 26

18 2.7 21.6 1.566 1.1 27

19 2.9 23.2 1.682 1.2 29

20 3.0 24.0 1.740 1.3 30

21 3.1 24.8 1.798 1.3 31

22 3.3 26.4 1.914 1.4 33

23 3.4 27.2 1.972 1.4 34

24 3.5 28.0 2.030 1.5 35


Reagent Volume Guide For various numbers of 24-well Inserts in a 24-well Plate (all volumes are in mL)

24-well Insert 10X MEM Collagen Neutralizing Solution Cells or Medium Total

x y z c

1 0.12 0.96 0.070 0.050 1.2

2 0.15 1.2 0.087 0.063 1.5

3 0.17 1.4 0.099 0.071 1.7

4 0.20 1.6 0.116 0.084 2.0

5 0.22 1.8 0.128 0.092 2.2

6 0.24 1.9 0.139 0.101 2.4

7 0.27 2.2 0.157 0.113 2.7

8 0.29 2.3 0.168 0.122 2.9

9 0.32 2.6 0.186 0.134 3.2

10 0.34 2.7 0.197 0.143 3.4

11 0.36 2.9 0.209 0.151 3.6

12 0.39 3.1 0.226 0.164 3.9

13 0.51 4.1 0.296 0.214 5.1

14 0.54 4.3 0.313 0.227 5.4

15 0.56 4.5 0.325 0.235 5.6

16 0.58 4.6 0.336 0.244 5.8

17 0.61 4.9 0.354 0.256 6.1

18 0.63 5.0 0.365 0.265 6.3

19 0.66 5.3 0.383 0.277 6.6

20 0.68 5.4 0.394 0.286 6.8

21 0.70 5.6 0.406 0.294 7.0

22 0.73 5.8 0.423 0.307 7.3

23 0.75 6.0 0.435 0.315 7.5

24 0.78 6.2 0.452 0.328 7.8


RAFT™ Kits Ordering Information

Additional information and online ordering available at: www.lonza.com/raft

Related Product Information

Clonetics™ Human and Animal Primary Cells: Visit www.lonza.com/primary for specific cell types and recommended culture medium

Cat. No. Description

016-1R16 RAFT™ Small Kit, 12 rxns, reagent & 24-well plate absorbers

016-1R18 RAFT™ Small Kit, 12 rxns, reagent & transwell insert absorbers

016-1R17 RAFT™ 96-well Small Kit, reagent and 1 x 96-well plate absorbers

016-1R10 RAFT™ 96-well Bundle Kit, contains (016-0R94 & 016-0R92)

016-1R24 RAFT™ 24-well Bundle Kit, contains (016-0R94 & 016-1R32)

016-1R25 RAFT™ 24-well Insert Bundle Kit (016-0R94 & 016-1R33)

016-1R32 RAFT™ Absorbers for 24-well plate, 48 quantity

016-1R33 RAFT™ Insert Absorbers for 24-well plate, 48 quantity, for inserts

016-0R92 RAFT™ Plate Kit, 4 × 96-well clear plate & 4 × 96-well absorber

016-0R94 RAFT™ Reagent Kit for 3D culture

Lonza Walkersville, Inc. – Walkersville, MD 21793

For research use only. Not for use in diagnostic procedures.

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