Scale-Up of Mammalian Cell Culture using a New Multilayered Flask

1. Protocol to use Multi-Flasks for cell culture

1. Preparation of vessels and adding cell suspension
   1. Prepare cell growth medium as needed.
   2. Line up Multi-Flask vertically on its side with the cap facing up on the work surface (sterile laminar flow hood).
      1. These flasks are available in 3 and 5-layer formats and use a similar work-flow pattern as a T-175 flask.
   3. Loosen and remove caps. Add required amount of pre-warmed medium into flask using a 50 or 100 mL pipette or by pouring.
      1. Pipettes that are ≤ 10 ml can reach the bottom of the vessel when Multi-Flasks are placed vertically with cap facing upwards. Pipettes ≥ 10ml can reach into the vessel immediately past the logo on the Multi-Flask, thereby providing a convenient portal to add larger volumes of media to vessel.
   4. To avoid bubbling of medium, allow liquid stream to flow along the inner wall of the Multi-Flask lid (logo-side).
   5. Add cell suspension from a concentrated cell stock into growth medium through the top layer using a 10 mL pipette and cap flasks.
      
      Tips:
      – Transport Multi-Flasks on a cart to incubator site and perform remaining steps.
      – The cell seeding density will vary depending on the cell type, medium and culture duration need. Begin with the seeding density and media volume to that used in standard T-175 flasks and multiply by 3 or 5 depending on the Multi-Flask format used.
2. Mixing of cells
   1. Mix position: Hold the Multi-Flask upright with the logo facing you and turn counter clockwise to a 45°angle with mix port facing you.
   2. Holding at the same angle, gently tilt Multi-Flask from front to back (neck away from you) until liquid in the top layer drains fully downwards through the mix port. Pivot on mix port-side.
   3. Likewise, gently rock Multi-Flask from back to front (neck towards you) until medium drains fully from the bottom layer towards the top through the mix port. Repeat Steps 1.2.2 and 1.2.3 one more time to ensure proper mixing.
   4. Bring Multi-Flask back to mix position (Step 1.2.1) and proceed to Step 1.3
   5. Alternatively, cell suspension can be prepared externally from the Multi-Flask and cell suspension can be added to the vessel using a pipette or by gentle pouring.
      1. Mix port allows in-vessel mixing of cells with media and eliminates the need to make large volumes of cell suspensions externally.
      2. It also allows media equalization across the layers of the Multi-Flask
3. Equilibration of fluid
   1. After mixing/adding of cell suspension, place Multi-Flask vertically on a flat work surface to equalize liquid volume equally in all the layers.
4. Partition liquid into each layer
   1. Hold the Multi-Flask with the logo facing you and turn clockwise to a 45°angle to partition the liquid into each of the layers.
      
      Tip:
      – For best results, it is important to use a flat work surface for Steps 1.3-1.4
5. Transportation
   1. Once media containing cell suspension is partitioned, transport Multi- Flask at the same 45° angle (clockwise) as in Step 1.4.1 with media away from the mix port into incubator. This position is also suitable when removing flasks from incubator for viewing on a microscope.
6. Placing Multi-Flask onto incubator shelf
   1. Holding Multi-Flask at the 45° angle (clockwise, away from mix-port), gently rotate it down horizontally onto the incubator surface (using the corner away from the mix port as a pivot). Lay Multi-Flask with Logo facing up.
      1. Falcon Multi-Flask design allows vessels to be stacked and holds them in place by a sturdy stacking rib.
7. Distribution of cells and reagents
   1. After placing Multi-Flask flat on work surface, gently rock back and forth and side-to side to distribute cells evenly onto culture surfaces taking care not to spill liquid from each layer. Stack flasks.
      1. This Flask is made of optically clear material and cells on the last layer can be easily viewed on a microscope.
8. Media exchange
   1. Aspirate while tilting the Multi-Flask to the left (mix port-side) with the logo facing you.
   2. Then tilt, the Multi-Flask to the right, continuing to aspirate all residual media.
   3. Add appropriate amount of media and follow Steps 1.3-1.7

2. Harvesting cells from Multi-Flasks

1. To dissociate cells, line up Multi-Flasks vertically and bring each one to Mix position (Step 1.2.1). Gently invert flasks with neck facing you so as to allow media to drain to the top layer of Multi-Flask.
2. Insert a 5 or 10 mL aspirating tip through the neck until you reach liquid level near the mix port. Aspirate exhausted medium.
3. Add dissociation reagent/wash buffer and bring to Mix position as in Step 1.2.1, then proceed and follow steps 1.3-1.7. Neutralize with growth medium /neutralizing agent and pour cell suspension into a receiving tube OR invert flask such that cells drain to the top layer of the vessel and collect cells using a 10 mL pipette.
   
   Tip: To enhance recovery of cells or reagents, bring Multi-Flask to mix position (Step 1.2.1), invert with neck towards operator to allow complete drainage of media from all layers to the top layer. Then, tilt Multi-Flask clockwise to 45° angle (away from the mix port) while the Multi-Flask remains inverted. Use a pipette (1-10 mL) to collect any remaining reagents.

3. Recommended working volume in Falcon Multi-Flasks

Growth media

3-Layer: 75-150 mL per Multi-Flask
5-Layer: 125-250 mL per Multi-Flask

Dissociation agent

3-Layer: ≥15 mL per Multi-Flask
5-Layer: ≥25 mL per Multi-Flask

Tip: Begin with medium volume used in standard T-175 flasks and multiply by 3 or 5 times depending on the Multi-Flask format evaluated so that mL per unit surface area remains the same.

4. Representative Results:

1. Design of Falcon Multi-Flask

Figure 1.: Multi-Flask Cell Culture vessels are available in a 3- and 5-layer stackable format for scale-up of cells providing 525 and 875 cm² growth surface area, respectively. Pipette access facilitates addition and removal of cells and reagents into-and out of the vessel. Presence of mix-port allows for rapid in-vessel mixing and equalization of media across all layers of the Multi-Flask.

2. Cell Yield using Multi-Flask:

These vessels are available in 3-layer and 5-layer formats which correspond to 3 and 5-times the surface area of T-175 flasks. Accordingly, ≥ 3 and 5 times (130± 6.8 x 10⁶ and 218 ± 23.6 x 10⁶ cells, respectively) the number of BHK-21 (baby hamster kidney) cells were grown and recovered from Multi-Flask compared to T-175 flasks (43.2 ± 3.5 x 10⁶ cells; Fig.2A). Cell yield per unit surface area was equivalent in 3- and 5-layer Multi-Flasks and T-175 flasks for BHK-21, LnCAP (human prostate adenocarcinoma cell line), Hep-G2 (human hepatocarcinoma cell line), EcoPack 2-293 (human kidney cell line) cultured for a period of 48-96h (Fig.2B) in growth media (35 ml per layer) as recommended by cell vendor (ATCC, Sigma and/or Clontech). Cells were enumerated on an automated Vi-CELL counter ⁽¹⁾.

Figure 2A : Three and five times the number of BHK-21 cells were grown and recovered from 3- and 5-layer Multi-Flasks compared to T-175 flasks. Expected yield was determined using mean cell yield from control T-175 flasks multiplied by three and five times for the 3- and 5-layer Multi-Flasks respectively (n=4 flasks/format).

Figure 2B : Cell yield per cm² was equivalent in 3- and 5-layer Multi-Flasks and T-175 flasks for BHK-21, LnCAP, HepG2 and EcoPack2-293 cells. Each bar represents mean of 4 to 6 flasks. BHK-21 cells (11,000 cells/cm2) were cultured for 72 hours, LnCAP cells (20,000 cells/cm2) and EcoPack2-293 cells (˜35,000 cells/cm2) were cultured for 96 hours and HepG2 cells (25,000 cells/cm²) were cultured for 48 hours prior to harvest.

3. Media distribution among layers of Multi-Flask

Cell culture medium (DMEM; Invitrogen) was added to 5-layer Multi-Flasks (250 mL/5-layer vessel) and partitioned into layers according to protocol described above. Media distribution was measured by drilling holes in each layer and media pumped out from individual layers. Weight of fluid recovered from each layer was found to be relatively uniform from layer to layer as shown in Fig 3. They are as follows: 51.8 ± 0.73, 50.3 ± 0.58, 50.21 ± 0.13, 49.88 ± 0.35, 49.45 ± 0.37 (gm).

Figure 3. Uniform media distribution in each of the five layers of a 5-layer Multi-Flask. Cell culture medium was added to Multi-Flasks (250 ml/5-layer vessel), equilibrated & partitioned to individual layers. The media was pumped out through holes drilled into individual layers and fluid weight was recorded from each layer (n=6 flasks).

4. Cell distribution between layers of Multi-Flask

Cells can be added and mixed within the Multi-Flask. We simulated distribution of cells between layers of Multi-Flask using beads (10μm; PolySciences Inc.) similar in size to cells. Bead suspension was added into Multi-Flask vessel using a 10 mL pipette through the top layer and mixed with media in the vessel as described in the protocol above. Bead distribution was measured by drilling holes in each layer and fluid containing bead suspension was pumped out from individual layers. Bead concentration recovered from each layer was read on a Coulter Counter and recorded. Shown below are equivalent inter-layer bead distributions in 3-layer Multi-Flasks (Fig.4A). The mix-port enables homogenous distribution of cells and reagents between Multi-Flask layers.

Figure 4A : Bead distribution in each of the three layers of a 3-layer Multi-Flask. A suspension of beads (3.6 x10⁶/ml) was added to medium dispensed into Multi-Flasks (bead suspension:media volume is 1:10, vol:vol) and mixed, followed by equilibration and partition steps using the protocol described. Bead concentration recovered from each layer (medium pumped through holes drilled on each layer) was read on a Coulter Counter and recorded. Shown below are equivalent inter-layer bead distributions in 3- layer Multi-Flasks (n=5 flasks)

Shown below are representative images of Ecopack 2-293 staining patterns of cells grown to >80% confluence on 3-layer Multi-Flasks in supplemented growth media. Cell monolayers were fixed and stained with crystal violet and Multi-Flask layers were then cut and images scanned ⁽²⁾. Note, cell patterning was homogenous on all layers of Multi-Flask (Fig.4B). Similar results were obtained with multiple cell types evaluated (data not shown).

Figure 4B : This figure illustrates homogeneous cell growth between layers of Multi-Flasks. Ecopack-2-293 cells grown to >80% confluence in 3-layer Multi-Flasks and T-175 were fixed and stained with crystal violet. Multi-Flask vessels were cut and each stained layer was scanned.

5. Air supply in Multi-Flask:

Analysis of spent media using BioProfile FLEX analyzer ^(3,4) (Nova Biomedical) from EcoPack2-293 cells cultured for 96 hours revealed no difference in air saturation of cells grown in 5-layer Multi-Flasks vs. T-175 flasks ( 81.03 ± 1.9 vs. 83.4 ± 5.8 % ambient O₂)_.

Figure 5 : Air saturation (% ambient O₂) of spent media were similar in pre-mixed media from 5-layer Multi-Flasks vs. T-175 flasks. EcoPack2-293 cells were seeded at a density of 35,000 cells/cm² and cultured for 96 hours prior to media analysis (n=3 flasks).