DELIVERY ACROSS CELL PLASMA MEMBRANES
Delivering a payload across a plasma membrane of a cell includes providing a population of cells and contacting the population of cells with a volume of an aqueous solution. The aqueous solution includes the payload and alcohol content greater than 5 percent concentration. The volume of the aqueous solution may be a function of exposed surface area of the population of cells, or may be a function of a number of cells in the population of cells. Related compositions, apparatus, systems, techniques, and articles are also described.
1 .- 99 . (canceled)
100 . An apparatus for delivering a payload across a plasma membrane of a cell, the apparatus comprising:
a pneumatic generator configured to produce gas under a pressure;
an atomizer operatively coupled to the pneumatic generator and capable of forming a spray;
a reservoir configured to contain an aqueous solution, the aqueous solution including the payload; and
a valve between the pneumatic generator and atomizer, the valve switchable between a closed position for preventing the gas under the pressure from activating the atomizer and an open position for allowing the gas under the pressure to activate the atomizer to produce the spray, the spray including colloidal droplets, the valve having a switching speed less than 250 milliseconds;
wherein the atomizer is oriented towards a support capable of supporting a population of cells for contacting the population of cells with a volume of the aqueous solution.
101 . The apparatus of claim 100 , wherein the volume is a function of: (i) exposed surface area of the population of cells; or (ii) a number of cells in the population of cells.
102 . The apparatus of claim 100 , wherein the volume is between 6.0×10 −7 microliter per cell and 7.4×10 −4 microliter per cell.
103 . The apparatus of claim 100 , wherein the volume is between 9.3×10 −6 microliter per cell and 2.8×10 −5 microliter per cell.
104 . The apparatus of claim 100 , wherein the volume is about 1.9×10 −5 microliters per cell, wherein about is within 10 percent.
105 . The apparatus of claim 100 , wherein the volume is between 4.9×10 −6 microliter per cell and 2.2×10 −3 microliter per cell.
106 . The apparatus of claim 100 , wherein the population of cells includes an exposed surface, and wherein the volume is between 2.6×10 −9 microliter per square micrometer of exposed surface area of the population of cells and 1.1×10 −6 microliter per square micrometer of exposed surface area of the population of cells.
107 . The apparatus of claim 100 , wherein the population of cells includes an exposed surface, and wherein the volume is between 5.3×10 −8 microliter per square micrometer of exposed surface area and 1.6×10 −7 microliter per square micrometer of exposed surface area.
108 . The apparatus of claim 100 , wherein the population of cells includes an exposed surface, and wherein the volume is about 1.1×10 −7 microliter per square micrometer of exposed surface area, wherein about is within 10 percent.
109 . The apparatus of claim 100 , wherein the apparatus is configured to deliver a total volume of aqueous solution of 20 μl to a cell-occupied area of about 1.9 cm 2 , wherein about is within 10 percent.
110 . The apparatus of claim 100 , wherein the apparatus is configured to deliver a total volume of aqueous solution of 10 μl to a cell-occupied area of about 0.95 cm 2 , wherein about is within 10 percent.
111 . The apparatus of claim 100 , wherein said colloidal droplets comprises discrete units of volume ranging in size from 30-100 μm in diameter.
112 . The apparatus of claim 100 , wherein said spray comprises discrete units of volume with a diameter of about 30-50 μm, wherein about is within 10 percent.
113 . The apparatus of claim 100 , wherein the atomizer, when the valve is in the open position, produces colloidal droplets having a diameter between 30 and 100 micrometers.
114 . The apparatus of claim 100 , wherein the atomizer, when the valve is in the open position, produces colloidal droplets having a diameter between 30 and 50 micrometers.
115 . The apparatus of claim 100 , wherein the switching speed is a time required for the valve to transition between the closed position and the open position.
116 . The apparatus of claim 100 , wherein the valve has a switching speed between 50 and 200 milliseconds.
117 . The apparatus of claim 100 , further comprising the population of cells, and wherein the population of cells is in contact with said aqueous solution for 0.1-10 minutes prior to adding a second volume of buffer or culture medium to submerse or suspend said population of cells.
118 . The apparatus of claim 117 , wherein the buffer or culture medium comprises phosphate buffered saline (PBS).
119 . The apparatus of claim 100 , further comprising the population of cells, wherein the population of cells is in contact with the aqueous solution for 2 seconds to 5 minutes prior to adding a second volume of buffer or culture medium to submerse or suspend the population of cells.
120 . The apparatus of claim 100 , further comprising the population of cells, wherein the population of cells is in contact with the aqueous solution for 30 seconds to 2 minutes prior to adding a second volume of buffer or culture medium to submerse or suspend the population of cells.
121 . The apparatus of claim 120 , further comprising the population of cells, wherein the population of cells is in contact with the aqueous solution for about 1-2 minutes prior to adding the second volume of buffer or culture medium to submerse or suspend the population of cells.
122 . The apparatus of claim 100 , further comprising the population of cells.
123 . The apparatus of claim 122 , wherein the population of cells comprises adherent cells.
124 . The apparatus of claim 123 , wherein the adherent cells include at least one of primary or immortalized mesenchymal stem cells, lung cells, neuronal cells, fibroblasts, human umbilical vein (HUVEC) cells, Chinese hamster ovary (CHO) cells, human embryonic kidney (HEK) cells, red blood cells, ovum cells.
125 . The apparatus of claim 122 , wherein the population of cells comprises non-adherent cells.
126 . The apparatus of claim 125 , wherein the non-adherent cells include at least one of primary or immortalized hematopoietic stem cell (HSC), T cells, natural killer (NK) cells, cytokine-induced killer (CIK) cells, human cord blood CD34+ cells, and B cells.
127 . The apparatus of claim 100 , wherein the population of cells is substantially confluent, wherein substantially is greater than 75 percent confluent.
128 . The apparatus of claim 100 , wherein the population of cells form a monolayer of cells.
129 . The apparatus of claim 100 , further comprising the aqueous solution.
130 . The apparatus of claim 129 , wherein the aqueous solution further comprises an alcohol at or greater than 2 percent concentration.
131 . The apparatus of claim 129 , wherein said aqueous solution comprises an ethanol concentration of 10 to 30%.
132 . The apparatus of claim 129 , wherein said aqueous solution comprises one or more of 75 to 98% H 2 O, 2 to 45% ethanol, 6 to 91 mM sucrose, 2 to 35 mM KCl, 2 to 35 mM ammonium acetate, and 1 to 14 mM (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) (HEPES).
133 . The apparatus of claim 129 , wherein the payload comprises a small chemical molecule, a peptide or protein, or a nucleic acid.
134 . The apparatus of claim 133 , wherein the small chemical molecule comprises a molecular mass less than 1,000 Da.
135 . The apparatus of claim 134 , wherein the chemical molecule comprises MitoTracker® Red CMXRos, propidium iodide, methotrexate, or DAPI (4′,6-diamidino-2-phenylindole).
136 . The apparatus of claim 133 , wherein the peptide comprises ecallantide, liraglutide, or icatibant.
137 . The apparatus of claim 133 , wherein the nucleic acid molecule comprises a small-interfering RNA (siRNA molecule).
138 . The apparatus of claim 137 , wherein the siRNA molecule comprises a molecular mass of 15,000 Da.
139 . The apparatus of claim 133 , wherein the protein comprises about a molecular mass of 1,000 to 500,000 Da.
140 . The apparatus of claim 133 , wherein the protein comprises an antibody or fragment thereof.
141 . The apparatus of claim 140 , wherein the antibody or fragment thereof comprises an anti-actin antibody, an anti-GAPDH antibody, an anti-Src antibody, an anti-Myc ab, and an anti-Raf antibody.
142 . The apparatus of claim 133 , wherein the nucleic acid molecule comprises a molecular mass of greater than 5,000,000 Da.
143 . The apparatus of claim 129 , wherein the payload comprises a therapeutic agent.
144 . The apparatus of claim 143 , wherein the therapeutic agent includes at least one of cisplatin, aspirin, a statin, and fluoxetine.
145 . The apparatus of claim 129 , wherein the payload comprises a diagnostic agent.
146 . The apparatus of claim 145 , wherein the diagnostic agent includes at least one of methylene blue, patent blue V, and indocyanine green.
147 . The apparatus of claim 129 , wherein the payload comprises a fluorescent molecule.
148 . The apparatus of claim 129 , wherein the payload comprises a detectable nanoparticle.
149 . The apparatus of claim 148 , wherein the nanoparticle comprises a quantum dot.
150 . The apparatus of claim 100 , wherein the support includes a vessel for containing the cell population, the apparatus further comprising:
a vacuum for removal of media from the vessel.
151 . The apparatus of claim 150 , wherein the vacuum is configured to remove media from the vessel via a bottom of the vessel.
152 . The apparatus of claim 150 , wherein the vacuum is configured to remove media from a top of the cell population.
153 . The apparatus of claim 100 , wherein the valve is capable of allowing continuous gas flow.
154 . The apparatus of claim 100 , wherein the support includes a vessel for containing the cell population, the vessel including a single well.
155 . The apparatus of claim 100 , wherein the support includes a vessel, the vessel including a filter.
156 . The apparatus of claim 155 , wherein the filter includes a three dimensional scaffold.
157 . The apparatus of claim 155 , wherein the filter includes a pore size of less than 10 micrometers.
158 . The apparatus of claim 100 , wherein the support include a biological membrane.
159 . The apparatus of claim 158 , wherein the biological membrane includes a biological tissue.
160 . The apparatus of claim 159 , wherein the biological tissue includes a skin tissue, a tracheal tissue, and/or a biological organ.
161 . The apparatus of claim 100 , wherein the support includes a synthetic membrane.
162 . The apparatus of claim 161 , wherein the synthetic membrane includes an aluminum membrane or a plastic membrane.
163 . The apparatus of claim 100 , wherein the support includes a vessel for containing the cell population, the vessel including a porous cell culture plate insert.
164 . The apparatus of claim 100 , wherein the atomizer includes an emitter configured to provide a conical spray zone.
165 . The apparatus of claim 100 , wherein the spray is emitted at a pressure between 0.5 bar and 2.5 bar.
166 . The apparatus of claim 165 , wherein the apparatus is configured to control the pressure.
167 . The apparatus of claim 100 , wherein the pneumatic generator includes a gas reservoir.
168 . The apparatus of claim 167 , wherein the gas reservoir includes a pressure head generator.
169 . The apparatus of claim 168 , wherein the pressure head generator is configured to compress gas in the gas reservoir to maintain the gas under a pressure.
170 . The apparatus of claim 100 , wherein the apparatus is configured to uniformly contact the aqueous solution with the population of cells using the spray.
171 . The apparatus of claim 100 , further comprising the aqueous solution, wherein the payload comprises molecules of more than one molecular weight.
172 . The apparatus of claim 100 , further comprising the aqueous solution, wherein the payload comprises a first molecule and a second molecule, the second molecule different from the first molecule.
173 . The apparatus of claim 100 , further comprising the population of cells, wherein the population of cells are attached to a support or scaffold.
174 . The apparatus of claim 100 , wherein the apparatus is configured to deliver a total volume of aqueous solution of 10 μl-100 μl.